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Studies on histone modification and chromatin structure in developing trout testi Honda, Barry M. 1975

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STUDIES ON HISTONE MODIFICATION AND CHROMATIN STRUCTURE IN DEVELOPING TROUT TESTIS by BARRY M. HONDA B . S c , McMaster U n i v e r s i t y , 1971 THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of B i o c h e m i s t r y F a c u l t y o f Medicine We accept t h i s t h e s i s as conforming t o the r e q u i r e d srta*id/ard 'S'ep-hember 1975 U n i v e r s i t y of B r i t i s h Columbia In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of "Bl^CM 6 M KTfcM The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Sep* /9 , / f ? S ABSTRACT During spermatogenesis i n rainbow t r o u t , a synchronous development of stem c e l l s •> spermatocytes -*• spermatids -*• mature sperm o c c u r s , w i t h replacement of the h i s t o n e s i n chromatin by protamines. PART A: Hi s t o n e m e t h y l a t i o n Along w i t h h i s t o n e a c e t y l a t i o n and p h o s p h o r y l a t i o n , methyl-a t i o n o f s p e c i f i c l y s y l r e s i d u e s o f h i s t o n e s H3 and H4 can be observed. T h i s h i s t o n e m e t h y l a t i o n o c c u r s predominantly i n the l a r g e d i p l o i d stem c e l l s and primary spermatocytes, which a c t i v e l y s y n t h e s i z e DNA and h i s t o n e s . In spermatids, h i s t o n e m e t h y l a t i o n i s minimal and so prob a b l y has no r o l e i n the r e -placement of h i s t o n e s by protamine. Other l a b e l l i n g e x p e r i -ments suggest t h a t h i s t o n e H4 m e t h y l a t i o n i s a l a t e event i n the c e l l c y c l e , o c c u r r i n g a f t e r the s y n t h e s i s , a c e t y l a t i o n and d e a c e t y l a t i o n o f h i s t o n e H4. T h i s m e t h y l a t i o n may be necessary f o r h i s t o n e p h o s p h o r y l a t i o n or chromatin condensation p r i o r to c e l l d i v i s i o n . PART B; Chromatin s u b u n i t s t r u c t u r e When a sample of t r o u t t e s t i s n u c l e i i s d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e , the DNA i s c l e a v e d almost e n t i r e l y t o d i s c r e t e fragments approximately 200 base p a i r s l o n g and mul-t i p l e s t h e r e o f . The same DNA fragments can be ob t a i n e d when i s o l a t e d chromatin, as opposed t o i n t a c t n u c l e i , i s nuclease - i t -d i g e s t e d . These DNA fragments can a l s o be found i n d i s c r e t e chromatin " s u b u n i t s " i s o l a t e d from n u c l e a s e - d i g e s t e d n u c l e i . Sedimentation through sucrose g r a d i e n t s , o r v e l o c i t y sedimen-t a t i o n i n an a n a l y t i c a l u l t r a e e n t r i f u g e s e p a r a t e s these chrom-a t i n s u b u n i t s i n t o l i s (monomer), 16S (dimer), 22S (trimer) e t c . s p e c i e s . Subunits can a l s o be f r a c t i o n a t e d on a Sepharose 2B column e q u i l i b r a t e d and run i n low s a l t . High s a l t (>40 mM NaCl) o r d i v a l e n t c a t i o n s (-5 mM) cause s u b u n i t p r e c i p i t a t i o n . Chromatin s u b u n i t s have a protein:DNA r a t i o o f a p p r o x i -mately 1.2 and c o n t a i n a l l the h i s t o n e s , i n c l u d i n g the t r o u t -s p e c i f i c h i s t o n e H6. There a re however no d e t e c t a b l e non-h i s t o n e chromosomal p r o t e i n s . M g + + p r e c i p i t a t e s o f the 11S chromatin monomers, when p e l l e t e d , a re t h i n and c l e a r , w h i l e oligomer Mg p e l l e t s are t h i c k and white. T h i s c o u l d r e f l e c t a more symmetrical or or d e r e d p a c k i n g o f 11S monomers, which are d e f i c i e n t i n h i s t o n e Hi. T h i s h i s t o n e may c r o s s l i n k the l a r g e r o l i g o m e r s , r e s u l t i n g i n a d i s o r d e r e d M g + + complex. These r e s u l t s are c o n s i s t e n t w i t h the s u b u n i t model o f chromatin s t r u c t u r e , based on 200 base p a i r long r e g i o n s o f DNA a s s o c i a t e d w i t h h i s t o n e s . These s u b u n i t s would be se p a r a t e d by n u c l e a s e - s e n s i t i v e DNA spacer r e g i o n s , and c r o s s l i n k e d by h i s t o n e H i . T e s t i s c o n s i s t i n g predominantly o f e a r l y spermatids ( m e i o t i c t i s s u e , c o n t a i n i n g mainly n u c l e o h i s t o n e ) g i v e s s i m i l a r y i e l d s o f DNA fragments and 11S s u b u n i t s . L a t e r stage t e s t i s (protamine has r e p l a c e d the h i s t o n e s ) however, g i v e s no DNA fragments o r 11S s u b u n i t s . T h i s presumably r e f l e c t s l a r g e d i f f e r e n c e s i n s t r u c t u r e between nucleoprotamine and n u c l e o -h i s t o n e . -iv-TABLE OF CONTENTS Page ABSTRACT i 1 TABLE OF CONTENTS ' iv LIST OF TABLES viii LIST OF FIGURES i% ACKNOWLEDGMENT xii PART A; HISTONE METHYLATION INTRODUCTION 1 I . The Histones 1 I I . Histone I n t e r a c t i o n s w i t h DNA and Other Histones 7 I I I . Covalent M o d i f i c a t i o n s of the Histones 9 IV. Spermatogenesis 15 MATERIALS AND METHODS 18 I. Chemicals and A b b r e v i a t i o n s 18 I I . I s o l a t i o n and C h a r a c t e r i z a t i o n of i n v i v o [ 1^C]methylated Trout T e s t i s Histones 19 (a) [ I l fC] methyl l a b e l l i n g of h i s t o n e s 19 (b) S t a r c h g e l e l e c t r o p h o r e s i s 20 (c) Determination of methylated amino a c i d s .. 20 (d) Determination of c e l l u l a r L-methionine and S-adenosyl-L-methionine 21 I I I . S i t e s of i n v i v o M e t h y l a t i o n of Trout T e s t i s Histones 22 (a) Automated p r o t e i n sequencing 22 (b) [ll*C)methyl peptides of h i s t o n e H3 22 (c) T r y p t i c peptides of [ ^ C l m e t h y l l a b e l l e d h i s t o n e H4 23 (d) Sequence determinations on [^C]methyl l a b e l l e d t r y p t i c peptides 24 -v-Page IV. Histone M e t h y l a t i o n i n D i f f e r e n t C e l l Types from Developing Trout T e s t i s 25 (a) C e l l separations 25 (b) Starch g e l e l e c t r o p h o r e s i s f o r hist o n e s .. 26 (c) Pulse-chase turnover experiments ......... 26 (d) R a d i o a c t i v i t y a n a l y s i s 27 V. K i n e t i c s of Methyl L a b e l l i n g of Histone H4 .... 27 (a) P r e p a r a t i o n of [ l l*C]methyl l a b e l l e d h i s t o n e H4 27 (b) R a d i o a c t i v i t y i n the modified species of his t o n e H4 28 RESULTS 29 I . I s o l a t i o n and C h a r a c t e r i z a t i o n of i n v i v o Methylated Trout T e s t i s Histones 29 (a) Which h i s t o n e s are methylated? 30 (b) Which methylated b a s i c amino a c i d s are present? 30 I I . S i t e s of i n v i v o M e t h y l a t i o n of L y s y l Residues Tn T e s t i s Histones 37 (a) Histone H3 37 (b) Histone H2B 45 (c) Histone H6 45 (d) Histone H4 47 I I I . (a) Histone M e t h y l a t i o n i n the D i f f e r e n t C e l l Types from Developing Trout T e s t i s .. 51 (b) Turnover of methyl groups on h i s t o n e l y s y l r e s i d u e s ^ 56 IV. K i n e t i c s of Histone H4 M e t h y l a t i o n 56 DISCUSSION 64 Me t h y l a t i o n S i t e s 64 Histone M e t h y l a t i o n i n D i f f e r e n t C e l l Types 66 Turnover of Histone Methyl Groups 66 K i n e t i c s of Histone H4 M e t h y l a t i o n 67 PART B: CHROMATIN SUBUNIT STRUCTURE INTRODUCTION 70 -vi-Page MATERIALS AND METHODS . .... 75 I . Chemicals and A b b r e v i a t i o n s 75 I I . P r e p a r a t i o n and Nuclease D i g e s t i o n of N u c l e i or I s o l a t e d Chromatin 75 I I I . Determination of Developmental Stages (histone:protamine r a t i o ) of Testes 76 IV. P r e p a r a t i o n of Chromatin Subunits from D i -gested N u c l e i 76 V. A n a l y s i s of DNA Fragments Produced by Nuclease D i g e s t i o n 77 (a) I s o l a t i o n of DNA fragments 77 (b) Q u a n t i t a t i o n of DNA 78 (c) Non-denaturing 2.5% polyacryalmide g e l e l e c t r o p h o r e s i s of DNA 78 (d) Denaturing 9 9% formamide, 6% p o l y a c r y l a -mide g e l s of DNA 79 VI. Sepharose 2B Chromatography of Chromatin Subunits 80 V I I . V e l o c i t y Sedimentation Experiments on Sub-u n i t s 80 V I I I . P r o t e i n Composition of Chromatin Subunits 82 (a) I s o l a t i o n of subunits from n u c l e i or whole chromatin 82 (b) Gel e l e c t r o p h o r e s i s of p r o t e i n s 83 (c) Q u a n t i t a t i o n of p r o t e i n s 84 RESULTS 86 I . C h a r a c t e r i z a t i o n of DNA Fragments Produced by M i c r o c o c c a l Nuclease D i g e s t i o n 86 (a) DNA fragments from d i g e s t e d n u c l e i 86 (b) DNA fragments from d i g e s t e d chromatin .... 92 I I . P r e p a r a t i o n of I s o l a t e d Chromatin Subunits .... 95 (a) Sucrose g r a d i e n t p r e p a r a t i o n of chromatin subunits 95 (b) Sedimentation v e l o c i t y a n a l y s i s of chromatin subunits 97 (c) Sepharose 2B chromatography 101 Page I I I . T r out T e s t i s Chromatin Subunit S t r u c t u r e : Comparison of Nu c l e o h i s t o n e and Nucleo-protamine 101 IV. P r o t e i n Composition of Chromatin Subunits 103 V. The Behaviour of Chromatin Subunits i n S a l t ... 107 DISCUSSION 110 DNA D i g e s t i o n P a t t e r n s 110 P r o t e i n Composition and Arrangement i n Chromatin Subunit P a r t i c l e s 112 Chromatin S t r u c t u r e During T e s t i s M a t u r a t i o n H 8 C o n c l u s i o n 119 BIBLIOGRAPHY . . . . 123 - v i i i -LIST OF TABLES Page PART A: HISTONE METHYLATION I. Nomenclature and p r o p e r t i e s o f h i s t o n e s 3 I I . Sequence da t a a v a i l a b l e f o r the h i s t o n e s 4 I I I . [ 1 !*C]methyl l a b e l l e d p e p t i d e s d e r i v e d from h i s t o n e H3 44 PART B; CHROMATIN SUBUNIT STRUCTURE I. Chromatin Su b u n i t s - S e d i m e n t a t i o n V e l o c i t y Data (a) Scanner O p t i c s 98 (b) S c h l i e r e n O p t i c s 99 I I . M i c r o c o c c a l Nuclease D i g e s t i o n on N u c l e o h i s t o n e and Nucleoprotamine 104 -ix-LIST OF FIGURES PART A; HISTONE METHYLATION  Fig u r e Page 1. L i C l g r a d i e n t s e p a r a t i o n of [ 1 "*C] methyl l a b e l l e d h i s t o n e s 31 2. Separation of [ J I*C]methyl h i s t o n e s from protamines. 32 3. P r e p a r a t i o n of [^C]methyl l a b e l l e d h i s t o n e f r a c -t i o n s on Bi o - G e l P-10 33 4. Presence of [ 1^C]methionine and [ ^ C l m e t h y l amino ac i d s i n the hist o n e s 35 5. Separation of e-N-methyl-lysines on a Technicon amino a c i d a n a l y s e r 36 6. Automated degradation of [ 1^C]methyl l a b e l l e d t e s t i s h i s t o n e H3 39 7. Recovery of a l a n i n e d u r i n g automated s e q u e n t i a l degradation of h i s t o n e H3 40 8. Sephadex G-50 chromatography of N-bromosuccinimide d e r i v e d peptides of [ C ] m e t h y l h i s t o n e H3 42 9. Sephadex G-10 chromatography of H3 t r y p t i c pep-t i d e s 43 10. Automated sequence a n a l y s i s of [ 1 4 C ] m e t h y l l a b -e l l e d t e s t i s h i s t o n e H2B 46 11. Sephadex G-25 chromatography of t r y p t i c peptides of maleylated [ l l*C]methyl h i s t o n e H4 48 12. Paper chromatography of t r y p t i c peptides of maleylated H4 49 13. Sequence a n a l y s i s of [^C]methyl h i s t o n e H4 50 14. The s i t e s of a c e t y l a t i o n and met h y l a t i o n of t e s t i s h i s t o n e s H3 and H4 52 15. I n c o r p o r a t i o n of L-[methyl- 3H]methionine and L - [ 1 ^ C ] l y s i n e i n t o d i f f e r e n t c e l l types from t r o u t t e s t i s 54 -x-F i g u r e Page 16. S y n t h e s i s and M e t h y l a t i o n o f h i s t o n e s i n the d i f f e r e n t c e l l types o f t r o u t t e s t i s 55 17. Turnover of [ 1 I +C] methyl groups i n t r o u t t e s t i s h i s t o n e s 57 18.. I n c o r p o r a t i o n of [ 3 H ] a r g i n i n e i n t o t e s t i s n u c l e i w i t h t i m e - c e l l v i a b i l i t y 58 19. The a c e t y l a t e d and pho s p h o r y l a t e d s p e c i e s o f h i s t o n e H4, separated by s t a r c h g e l e l e c t r o -p h o r e s i s 60 20. [ 3 H ] l y s i n e i n c o r p o r a t i o n i n t o h i s t o n e H4 w i t h time, as d e s c r i b e d by L o u i e and Dixon (84) 61 21. [ ^ C ] m e t h y l i n c o r p o r a t i o n i n t o h i s t o n e H4 as a f u n c t i o n of time 63 PART B: CHROMATIN SUBUNIT STRUCTURE 1. Scans of DNA from h i s t o n e and protamine stage n u c l e i separated on 2.5% p o l y a c r y l a m i d e g e l s 88 2. P l o t o f DNA band number v e r s u s square r o o t o f band m o b i l i t y f o r DNA se p a r a t e d on 2.5% p o l y a c r y l -amide g e l s 89 3. Denaturing 99% formamide, 6% p o l y a c r y l a m i d e g e l s e p a r a t i o n o f DNA fragments 90 4. C a l i b r a t i o n o f m o b i l i t y v e r s u s s i n g l e s t r a n d e d l e n g t h f o r n u c l e i c a c i d s separated on formamide g e l s 91 5. C h a r a c t e r i z a t i o n o f DNA fragments from i n t a c t n u c l e i and i s o l a t e d chromatin 93 6. 2.5% p o l y a c r y l a m i d e g e l s e p a r a t i o n o f DNA e x t r a c t e d from chromatin s u b u n i t monomers—minor bands 94 7. C h a r a c t e r i z a t i o n o f DNA i n chromatin s u b u n i t s a c r o s s a sucrose g r a d i e n t 96 8. Sedimentation v e l o c i t y a n a l y s i s o f chromatin from d i g e s t e d n u c l e i 100 -xi-F i g u r e Page 9. Sepharose 2B chromatography of chromatin i s o -l a t e d from n u c l e a s e - d i g e s t e d n u c l e i 102 10. H i s t o n e c o n t e n t i n chromatin s u b u n i t s a c r o s s a sucrose g r a d i e n t 106 11. P r e c i p i t a t i o n of chromatin s u b u n i t s by monovalent and d i v a l e n t c a t i o n s 108 - x i i -ACKNOWLEDGMENT I wish to thank Dr. Gordon H. Dixon f o r h i s i n i t i a l guidance and support during these s t u d i e s . A l s o , those f a c u l t y members who at one time or another served on my advisory committee — Drs. P.D. Bragg, Michael Smith, Gordon Tener, S.H. Zbarsky — gave me great help and super-v i s i o n . I owe a great debt to my present a d v i s o r , Dr. Peter Candido, f o r h i s comments, encouragement, e n t h u s i a s t i c i n t e r e s t and f r i e n d s h i p during the course of t h i s work. I a l s o wish to thank the many people i n the department who gave me advice and help, e s p e c i a l l y Drs. Stewart Gilmour, Andrew Louie, Ian G i l l a m , S h i r l e y G i l l a m , and my c o l l a b -o r a t o r and i n s p i r a t i o n f o r much of the l a t e r work, Dr. David B a i l l i e . Joe Durgo r e q u i r e s s p e c i a l mention f o r singlehandedly keeping the l a b o r a t o r y instruments working f o r us. L a s t l y , I should acknowledge the four years of support from the N a t i o n a l Research C o u n c i l of Canada, from whom I re c e i v e d a Centennial Science S c h o l a r s h i p . PART A: HISTONE IffiTHYLATION - 1 -INTRODUCTION I. The H i s t o n e s The h i s t o n e s a re b a s i c p r o t e i n s a s s o c i a t e d w i t h DNA i n the chromosomes o f e u k a r y o t i c organisms ( f o r reviews, see r e f s . 1-4). Miescher and l a t e r K o s s e l (5) f i r s t i s o l a t e d and d e s c r i b e d the h i s t o n e s , but f u r t h e r c h a r a c t e r i z a t i o n o f these p r o t e i n s was hampered by t h e i r apparent h e t e r o g e n e i t y . With improved tech n i q u e s f o r h i s t o n e f r a c t i o n a t i o n and a n a l y s i s i t i s now r e c o g n i z e d t h a t t h e r e a r e f i v e major s p e c i e s o f h i s t o n e i d e n t i f i a b l e by amino a c i d c o m p o s i t i o n , sequence, and e l e c t r o p h o r e t i c and chromatographic p r o p e r t i e s . These p r o t e i n s , p r e s e n t i n approximately e q u a l weight w i t h DNA, are g e n e r a l l y removed from the DNA by e x t r a c t i o n w i t h a c i d o r s a l t s o l u t i o n . R e c e n t l y the same 5 h i s t o n e types were des-c r i b e d from Neurospora c r a s s a (6 ) ; H i and H3 have not been found i n y e a s t , Saccharomyces c e r e v i s i a e (7), but p r o t e o l y s i s c o u l d account f o r t h i s r e s u l t (6). Hence i t i s l i k e l y t h a t , c o n t r a r y t o e a r l i e r r e p o r t s (8), the n u c l e a r b a s i c p r o t e i n s and chromatin s t r u c t u r e i n f u n g i are s i m i l a r t o those o f h i g h -e r eukaryotes. A g i v e n h i s t o n e type may show some h e t e r o g e n e i t y due t o the presence of minor components which v a r y i n sequence, o r to changes i n charge f o l l o w i n g c o v a l e n t m o d i f i c a t i o n o f amino a c i d s i d e c h a i n s . In a d d i t i o n , n u c l e a t e d e r y t h r o c y t e s ( i n b i r d s , f i s h , amphibia, r e p t i l e s ) c o n t a i n a s p e c i a l h i s t o n e , H5 (4), and rainbow t r o u t t i s s u e s have an e x t r a h i s t o n e H6 (9 ) . - 2 -The sperm c e l l s o f many animal s p e c i e s c o n t a i n s m a l l h i g h l y b a s i c "protamines" which may r e p l a c e the h i s t o n e s i n chromatin (10) . Table I p r e s e n t s the nomenclature and some p r o p e r t i e s o f the h i s t o n e s from c a l f thymus and ot h e r s o u r c e s . T h i s t h e s i s w i l l use the Hi t o H6 nomenclature proposed a t the CIBA Foundation meetings i n London, 1974 (11) . T a b l e I I summarizes the sources o f amino a c i d sequence data a v a i l a b l e f o r the h i s t o n e s . Of the f i v e major s p e c i e s , o n l y H i has not been co m p l e t e l y sequenced; however, a d j o i n i n g h a l v e s o f H i from two d i f f e r e n t s p e c i e s have been sequenced. Noteworthy f e a t u r e s o f the sequence d a t a are ( i ) the c l u s t e r -i n g o f b a s i c r e s i d u e s (and c o v a l e n t m o d i f i c a t i o n s ) t o s p e c i f i c p o r t i o n s o f a h i s t o n e molecule and ( i i ) the s t r i k i n g con-s e r v a t i o n o f amino a c i d sequence a c r o s s a p h y l o g e n e t i c t r e e . H i s t o n e s H3 and H4 are v e r y s i m i l a r i n terms o f a r g i n i n e content, extreme sequence c o n s e r v a t i o n , NH2-terminal c l u s t e r -i n g o f b a s i c amino a c i d s , s a l t e l u t i o n from DNA, and exte n -s i v e e-N-methylation o f s p e c i f i c l y s y l r e s i d u e s . More r e c e n t l y , H3 and H4 have been found a s s o c i a t e d as tetr a m e r s (32-34) . H4 has a pho s p h o r y l a t e d NH2-terminal s e r i n e (35) and c o n t a i n s e-N-mono- and d i m e t h y l - l y s i n e a t p o s i t i o n 20 (except f o r pea H4) and e - N - a c e t y l - l y s i n e a t p o s i t i o n 16 (12) and p o s i t i o n s 5 ,8 ,12 (36) . The presence of 3 - p h o s p h o h i s t i d i n e i n H4 has r e c e n t l y been r e p o r t e d (37) . H i s t o n e H3 c o n t a i n s e-N-mono-/ d i - and sometimes t r i m e t h y l - l y s i n e a t r e s i d u e s 9,27 (17-20) -3-TABLE I His t o n e A l t e r n a t e Nomenclatures M o l e c u l a r Weight NH 2-terminus HI H2A H2B H3 H4 H5 I, F l , KAP, l y s i n e - r i c h 21,000 I l b i , F2a2, LAK) s l i g h t l y 14,000 '' l y s i n e l i b * , F2b, KAS ) r i c h 13,800 I I I , F3, ARE ^ a r g i n i n e 15,300 IV, F 2 a l , GRK J r i c h 11,300 V, F2c, KSA, e r y t h r o c y t e - %20,000 s p e c i f i c A c e t y l - S e r Acety'l-Ser Pro A l a A c e t y l - S e r Thr H6 T, AKP, t r o u t - s p e c i f i c ^14,500 Pro -4-TABLE I I Histone Source* Extent of Sequence Reference H4 c a l f thymus complete 12,13 p i g complete 14 pea complete 15 r a t complete 16 H3 c a l f thymus complete 17 chicken complete 18 pea complete 19 carp complete 20 mollusc,cycad, r e s i d u e s 1 - 4 0 21 shark, sea u r c h i n t r o u t t e s t i s r e s i d u e s 1 - 2 5 22 H2B c a l f thymus complete 23 t r o u t t e s t i s r e s i d u e s 1 - 2 2 22 H2A c a l f thymus complete 24 t r o u t t e s t i s p a r t i a l (missing 2 i n t e r n a l sequences) 25 HI r a b b i t thymus res i d u e s 1 - 107 26,27 c a l f thymus res i d u e s 1 - 7 2 26 t r o u t t e s t i s r e s i d u e s 108 - 210 28 H5 chicken e r y t h r o c y t e r e s i d u e s 1 - 7 0 25 i n t e r n a l 29,30 H6 t r o u t t e s t i s r e s i d u e s 1 - 29 31 * P a r t i a l sequences from a few other sources, i n c l u d i n g t r o u t t e s t i s , are a v a i l a b l e f o r H4 (see r e f . 1 ). -5-and to a lesser extent at residues 4 (21) and 36 (13,21); £-N-acetyl-lysine has been l o c a l i z e d at residues 14,23 (17) and less at residues 9,18 (22). Low l e v e l s of H3 phosphoryla-t i o n may occur at s e r y l residues 10 and 28 (38). It i s i n t e r -esting that sequence microheterogeneity has been reported for pea histone H3, where at residue 96, 60% of molecules con-t a i n alanine and 40% have serine (19). Multiple components also occur for histones HI (26) and H5 (47) . Whether more examples of t h i s microheterogeneity can occur i n the histones i s s t i l l unresolved (e.g. r e f s . 19,39,48). Histones H2A and H2B also have c l u s t e r i n g of basic amino acids at the NH 2-terminus, but d i f f e r from H3, H4 i n d i s s o c i a t i n g from DNA at a lower s a l t concentration and i n having less highly conserved amino acid sequences. Dimers between these two histones have been reported (33,34). Trout t e a t i s H2A has an NH2-terminal sequence homologous to that of H4, and i s phosphorylated at the NH2-terminal serine (35) and acetylated at l y s i n e 5 (40). T e s t i s H2B i s phosphorylated (to low levels) at serine 6 (35) and i s acetylated at l y s y l residues 5,10,13 and 18 (22) . Histone HI d i f f e r s from the other major histone species i n i t s larger s i z e , stoichiometry [half the amount of any other histone (41)], heterogeneity within a given tissue (26), charge d i s t r i b u t i o n (NH2 and carboxyl-terminal regions are basic, middle i s hydrophobic), d i s s o c i a t i o n from DNA at low s a l t concentrations, and i t s increased evolutionary v a r i a b i l i t y , -6-seen v i a e l e c t r o p h o r e t i c s t u d i e s (42). The c a r b o x y l - t e r m i n a l sequence reporte d f o r t r o u t t e s t i s (28) i s i n t e r e s t i n g i n t h a t a l a r g e number of r e p e a t i n g sequences can be found — ( i ) 3 t e t r a p e p t i d e sequences -Lys-Ser-Pro-Lys-, each p o t e n t i a l l y phosphorylated, and ( i i ) s i x hexapeptide sequences a l l de-r i v a b l e from an a r c h e t y p a l sequence -V a l - A l a - A l a - L y s - L y s - P r o - . This r e s u l t s t r o n g l y suggests t h a t l a r g e p o r t i o n s of the molecule arose through repeated p a r t i a l gene d u p l i c a t i o n (28). H i has been reported t o c o n t a i n e-N-phospholysine (37). How-ever, aside from the NH2-terminal a c e t y l - s e r i n e , no a c e t y l a -t i o n or m e t h y l a t i o n of t h i s h i s t o n e has been observed, except perhaps f o r t r a c e amounts of N^-methyl-histidine (43). Histone H6 i s present i n low amounts (1% of t o t a l h i s -tone) i n t r o u t t i s s u e s (9). I t i s s i m i l a r to H i i n e l u t -a b i l i t y from chromatin and l y s i n e , a l a n i n e content, but i s l i k e H2B i n s i z e and l y s i n e r a r g i n i n e r a t i o . There i s as yet no known r o l e f o r t h i s h i s t o n e i n t r o u t t i s s u e s . I t i s now g e n e r a l l y b e l i e v e d t h a t the h i s t o n e s are syn-t h e s i z e d during S phase, on s m a l l polyribosomes i n the c y t o -plasm (44-46). The i s o l a t i o n of h i s t o n e mRNA t r a n s c r i p t s (49) which l i k e l y do not c o n t a i n 3' poly A (50) has l e d to the demonstration of m u l t i p l e (500-1000) copies of the h i s t o n e genes (51,52) i n sea u r c h i n , the l o c a l i z a t i o n of h i s t o n e genes by i n s i t u h y b r i d i z a t i o n to Drosophila melanogaster polytene -7-chromosomes (53) , and the r e c e n t i s o l a t i o n o f h i s t o n e genes i n c l o s e d c i r c u l a r p l asmid DNA (54). I t i s thought t h a t the t r a n s l a t i o n o f h i s t o n e s i s i n i t i a t e d w i t h methionine (57), r a t h e r than N - a c e t y l - s e r i n e (58), a l t h o u g h t h i s problem i s s t i l l not r e s o l v e d (see r e f . 28, d i s c u s s i o n ) . F o l l o w i n g t h e i r s y n t h e s i s , h i s t o n e s are t r a n s p o r t e d to the nucleus where they b i n d to DNA. Once bound to DNA, h i s t o n e s appear to be s t a b l e s t r u c t u r a l components o f chromatin, j u d g i n g from the s i m i l a r h a l f - l i v e s of DNA and h i s t o n e s i n p u l s e - c h a s e experiments (55,56). I I . H i s t o n e I n t e r a c t i o n s w i t h DNA and Other H i s t o n e s The nature o f histone-DNA i n t e r a c t i o n s i s s t i l l u n c l e a r , although the asymmetric charge d i s t r i b u t i o n o f h i s t o n e s , r e -v e a l e d by sequence s t u d i e s , l e d to the s u g g e s t i o n (15,17,23) t h a t the b a s i c r e g i o n s o f h i s t o n e s c o u l d i n t e r a c t w i t h DNA, w h i l e the n e u t r a l , hydrophobic r e g i o n s c o u l d b i n d o t h e r p r o t e i n s . A v a r i e t y of p h y s i c a l t e c h n i q u e s - n u c l e a r magnetic r e s -onance (NMR), c i r c u l a r d i c h r o i s m (CD), o p t i c a l r o t a t o r y d i s -p e r s i o n (ORD) - have been a p p l i e d to i s o l a t e d h i s t o n e s and h i s t o n e fragments i n s o l u t i o n ( f o r reviews see 1-4,59). An i n c r e a s e d o r d e r i n g of h i s t o n e s t r u c t u r e (more a - h e l i c a l regions) can be observed w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n s . Another approach to s t r u c t u r e i s to perform t h e o r e t i c a l c a l c u l a t i o n s (60) on the p r o b a b i l i t y o f h e l i x f o r m a t i o n i n the h i s t o n e s , along w i t h model b u i l d i n g . So, f o r example, Louie e t a l . (62), Sung and Dixon (61) and S h i h and Bonner (63) have b u i l t -8-a-helices for the NH2-terminal regions of H2A, H2B, H3 and H4, which f i t i n the major groove with l y s y l and a r g i n y l side chains i n appropriate positions to i n t e r a c t with DNA phos-phates. Histone-histone interactions (long considered an a r t i f a c t of i s o l a t i o n procedures) can be observed when "gentle" i s o -l a t i o n procedures ( i . e . not acid extraction) are used. Thus i t has been reported that H2A and H2B can i n t e r a c t as dimers (33,34), while H3 and H4 may associate as (H3)2 (H4) 2 t e t r a -mers (32-34). Bradbury, Crane-Robinson and co-workers have NMR evidence that the H3, H4 i n t e r a c t i o n i n s o l u t i o n occurs at the hydrophobic, carboxyl-terminal ends of the molecules (64) . How do the histones i n t e r a c t with DNA? Early experiments (65) on s a l t - t r e a t e d chromatin showed that the histones eluted in an order HI, then H2A, H2B, then H3, H4. This suggests an ordering for the "tightness" of i n t e r a c t i o n with DNA of H3, H4 > H2A, H2B > Hi. Indeed, removal of Hi r e s u l t s i n n e g l i g i b l e change i n nucleohistone conformation as character-ized by X-ray d i f f r a c t i o n , CD and ORD data (66-68). So, while H2A, H2B, H3 and H4 are necessary to maintain basic chromatin structure, Hi may be necessary as a " c r o s s l i n k i n g " molecule for chromosome c o i l i n g (69-71) and/or condensation. Very recent experiments, u t i l i z i n g nuclease digestion of chromatin i n i n t a c t n u c l e i , indicate that there i s an ordered, r e p e t i t i v e association of histones with DNA. These r e s u l t s -9-picture chromatin consisting l a r g e l y of 200 base p a i r long regions of DNA, associated with (probably) eight histone molecules. (See Part B of t h i s thesis.) The close association and stoichiometry of histones and DNA led to the idea that histones might be s p e c i f i c repres-sors c o n t r o l l i n g gene expression (72). I t i s now thought that there are too few d i s t i n c t histone types to account f o r the v a r i a t i o n i n gene expression seen i n higher organisms; histones may instead be s t r u c t u r a l components of chromatin and/or "coarse" or non-specific repressors of gene expression (104). The covalent modifications — acetylation, phosphoryla-t i o n , methylation etc. — of the histones may serve to modu-late histone-DNA or histone-histone i n t e r a c t i o n s , and hence allow changes i n the structure or function of chromatin. I I I . Covalent Modifications of the Histones Histone Acetylation As noted e a r l i e r i n the introduction, e-N-acetylated l y s y l residues occur i n the basic, NH2-terminal regions of histones H2A, H2B, H3 and H4. This a c e t y l a t i o n i s a post-synthetic (73), enzymatic (74-76) process, with a c e t y l co-enzyme A as acetate donor. Turnover of e-N-acetyl groups has been demonstrated (77,78) and histone deacetylases have been reported from c a l f thymus (79,80). Assuming the basic regions of histones i n t e r a c t with DNA, acetylation of l y s y l residues i n these regions eliminates -10-p o s i t i v e charges. T h i s c o u l d a l t e r h i s t o n e conformation and modulate the b i n d i n g o f h i s t o n e s to DNA, e i t h e r f o r gene ac-t i v a t i o n or chromatin assembly. That a c e t y l a t i o n o f a h i s t o n e can a l t e r i t s i n t e r a c t i o n w i t h DNA was r e c e n t l y r e p o r t e d by A d l e r e t a l . (81) who showed t h a t mono-acetylated H4 i s l e s s e f f e c t i v e than H4 i n changing DNA conformation, monitored by c i r c u l a r d i c h r o i s m . There are many r e p o r t s which c o r r e l a t e h i s t o n e a c e t y l a -t i o n and i n c r e a s e d gene a c t i v a t i o n : - i n phytohemagglutinin-t r e a t e d lymphocytes (82), r e g e n e r a t i n g r a t l i v e r (77) , and v a r i o u s c e l l types t r e a t e d w i t h hormones (83). However no d i r e c t c a u s e - e f f e c t r e l a t i o n s h i p i s e v i d e n t ; s i n c e t h e r e are probably too few h i s t o n e s p e c i e s to account f o r the complexity of gene e x p r e s s i o n i n h i g h e r organisms, a c e t y l a t i o n may " l o o s e n " the h i s t o n e s making the DNA a v a i l a b l e t o o t h e r molecules and s p e c i f i c gene a c t i v a t i o n . In t r o u t t e s t i s , two o t h e r p o s s i b l e f u n c t i o n s o f h i s t o n e a c e t y l a t i o n have been proposed. In spermatid c e l l s , h i s t o n e a c e t y l a t i o n may be r e q u i r e d to " l o o s e n " h i s t o n e s p r i o r t o t h e i r replacement by protamine (78). H i s t o n e a c e t y l a t i o n a l s o o c c u r s , a s s o c i a t e d w i t h DNA and h i s t o n e s y n t h e s i s , i n the l a r g e d i p l o i d stem c e l l s and primary spermatocytes (78) . Louie and Dixon (84) o b t a i n e d evidence t h a t a f t e r h i s t o n e H4 syn-t h e s i s , t h e r e was a r a p i d , o b l i g a t o r y d i a c e t y l a t i o n of H4, f o l l o w e d by a slower a c e t y l a t i o n (to the t e t r a a c e t y l a t e d form) and d e a c e t y l a t i o n (to the unmodified form). They h y p o t h e s i z e d -11-t h a t such a c e t y l a t i o n , by n e u t r a l i z i n g p o s i t i v e charges i n H4, allowed the c o r r e c t conformation f o r b i n d i n g t o DNA. Histo n e P h o s p h o r y l a t i o n The O-phosphorylation of s e r y l r e s i d u e s i n h i s t o n e s i s a l s o a p o s t s y n t h e t i c , enzymatic (85) pr o c e s s u s i n g ATP as phosphate donor. S e v e r a l h i s t o n e k i n a s e s , e i t h e r cAMP depen-dent o r independent, have been i s o l a t e d from n u c l e i o r c y t o -plasm (1-4). However, the p h y s i o l o g i c a l s i g n i f i c a n c e o f some of these enzymes i s u n c l e a r , s i n c e they are o f t e n c y t o p l a s m i c and s i n c e i s o l a t e d h i s t o n e s a re o f t e n n o n - s p e c i f i c s u b s t r a t e s f o r many p r o t e i n k i n a s e s . More r e c e n t l y , the p h o s p h o r y l a t i o n of l y s y l (HI) and h i s t i d y l (H4) r e s i d u e s by a phosphokinase from carcinosarcoma c e l l s has been r e p o r t e d (37). T h i s h i s t o n e p h o s p h o r y l a t i o n i s a dynamic p r o c e s s , w i t h phosphate t u r n o v e r and v a r i o u s h i s t o n e phosphatases r e p o r t e d (86 ). P h o s p h o r y l a t i o n may serve d i f f e r e n t f u n c t i o n s f o r the d i f f e r e n t h i s t o n e s . For h i s t o n e H i , t h e r e appear t o be two ki n d s o f p h o s p h o r y l a t i o n : ( i ) a p h o s p h o r y l a t i o n o f almost a l l H i molecules from S phase through t o m i t o s i s which c o r r e l a t e s w i t h chromosome condensation and m i t o s i s (62,70,71,87-89) and ( i i ) lower l e v e l s (a few percent) o f Hi p h o s p h o r y l a t i o n i n the response o f c e l l s t o hormones (90,91) and r e s u l t a n t gene a c t i v a t i o n . The k i n e t i c s o f h i s t o n e H2A p h o s p h o r y l a t i o n i n d i c a t e t h a t a r a p i d , o b l i g a t o r y p h o s p h o r y l a t i o n may be nece s s a r y f o r the c o r r e c t b i n d i n g o f H2A to DNA (35). A s i m i l a r model has been - 1 2 -proposed for the binding of protamines to DNA by Louie & Dixon (92). No function i s evident f o r the phosphorylation of histones H2B, H3 and H4. I t was o r i g i n a l l y thought that histone phosphorylation was involved i n the "loosening" of histones from DNA during t h e i r replacement by protamines. However, histone phosphorylation does not occur to a s i g n i f -icant extent i n spermatids (93,94), where t h i s replacement process occurs. Poly-ADP-ribosylation Poly (ADP-ribose) i s a polymer synthesized from NAD by a nuclear enzyme which joins ADP-ribose units i n a l " - 2 ' ribose-ribose linkage (for reviews, see 95,96). The structure can be represented as: n[Adenine-ribose-P-P-ribose-nicotinamide], n £ 30 Adenine I Ribose I P-ADP-ribose P—Ribose Adenine I Ribose I P -P—Ribose Adenine I Ribose I P P—Ribose T PD GH PD GH PD where venom phosphodiesterase cleaves at PD and a glycohydro-lase (97) cleaves at GH. This polymer i s covalently bonded to the histones i n an a l k a l i - l a b i l e linkage, possibly v i a a serine-phosphate i n histone Hi (98) . Further l o c a l i z a t i o n -13-of poly (ADP-ribose) may proceed following the preparation of s p e c i f i c antibodies against t h i s polymer (99). The function of t h i s polymer i n the nucleus i s unknown; i t may regulate DNA synthesis, chromatin structure or couple NAD and energy metabolism to DNA biosynthesis (95,96). Protein Methylation Like a c e t y l a t i o n and phosphorylation, the methylation of proteins i s a postsynthetic, enzymatic process u t i l i z i n g S-adenosyl-L-methionine as methyl donor (100). This methyla-t i o n occurs on c e r t a i n amino acids i n the protein backbone — x N -methyl-histidme; e-N-mono-, d i - and tr i m e t h y l - l y s i n e ; Q N -mono- and two dimethyl-arginines, and a few other minor amino acids (100). The carboxyl groups of aspartic and glutamic a c i d can also be found methylated as unstable methyl esters. Three d i f f e r e n t classes of protein methylases have been reported (100):- protein methylase I, a cytosol enzyme which methylates arginine; methylase I I , a p u r i f i e d cytosol enzyme which methylates glutamic and aspartic carboxyls; and methylase I I I , a nuclear enzyme which methylates l y s i n e . S o l u b i l i z a t i o n of the l a s t enzyme from chick n u c l e i has recently been re-ported (101). So far only c e r t a i n proteins of sp e c i a l function have been found methylated; the function of t h i s methylation i s unknown. Methylation of the basic amino acids increases the b a s i c i t y and hydrophobicity of the protein (100); modification -14-o f c a r b o x y l s l e a d s t o l o s s o f a n e g a t i v e c h a r g e . I t was e a r l i e r t h o u g h t t h a t m e t h y l a t i o n m i g h t p r o t e c t p r o t e i n s f rom d e g r a d a t i o n (100) ; however , more r e c e n t e x p e r i m e n t s on t h e p r o t e a s e 1 r e s i s t a n c e o f c h e m i c a l l y m e t h y l a t e d p r o t e i n s i n d i c a t e t h a t t h i s i s p r o b a b l y n o t t r u e (102) . In the case o f r h o d o p -s i n (103) , cytochrome c (100) and m y e l i n b a s i c p r o t e i n (104) , m e t h y l a t i o n may i n c r e a s e p r o t e i n h y d r o p h o b i c i t y f o r subsequent membrane i n t e r a c t i o n s . A c t i n , m y o s i n and some b a c t e r i a l f l a g e l l a r p r o t e i n s a r e a l s o m e t h y l a t e d (100) — t h e i r r o l e i n the c o n t r a c t i l e p r o c e s s i s unknown, though f u n c t i o n a l and d e v e l o p m e n t a l (105) d i f f e r e n c e s i n m y o s i n m e t h y l a t i o n have been r e p o r t e d . I n the c a s e o f t h e h i s t o n e s , DeLange, S m i t h and c o w o r k e r s , and o t h e r s , have i d e n t i f i e d s i t e s o f e - N - m e t h y l a t i o n o f s p e c -i f i c l y s y l r e s i d u e s i n h i s t o n e s H3 and H4 (12 -21 ) . I t i s n o t e -worthy t h a t t h e r e a r e d i f f e r e n c e s i n t h e e x t e n t o f m e t h y l a -t i o n a t t h e s e l y s y l r e s i d u e s , t h e most d r a m a t i c b e i n g t h e complete absence o f m e t h y l - l y s i n e i n pea H4 (15) . T h i s l a c k o f pea H4 m e t h y l a t i o n s u g g e s t s t h a t two enzymes, r e c o g n i z i n g H3 o r H4 m i g h t be p r e s e n t . Pea t i s s u e would l a c k t h e H4 r e c o g n i z i n g enzyme. P a t t e r s o n and D a v i e s have i n d e e d i s o l a t e d a pea h i s t o n e m e t h y l a s e w h i c h m e t h y l a t e s H3 b u t n o t H4 (106) . T h e r e a r e r e p o r t s t h a t o t h e r m e t h y l - a m i n o a c i d s a r e f o u n d i n G T h i s t o n e s (100) — N - m e t h y l - a r g i n i n e s and N - m e t h y l - h i s t i d i n e — b u t t h e l o c a l i z a t i o n , g e n e r a l i t y , and s i g n i f i c a n c e o f t h e s e a r e u n c l e a r . S i m i l a r l y , r e p o r t s t h a t h i s t o n e s H2A and H2B -15-are methylated (100,107) could a r i s e from contamination of these h i s t o n e s (incomplete h i s t o n e f r a c t i o n a t i o n e t c . ) . There are accounts of h i s t o n e m e t h y l a t i o n as a s t a b l e process (108), w h i l e others show i t to be a dynamic event, w i t h methyl groups t u r n i n g over during the c e l l c y c l e (107, 109). Histone m e t h y l a t i o n appears t o be a l a t e event i n the Hela c e l l c y c l e , o c c u r r i n g i n l a t e S and G 2 a f t e r DNA and h i s t o n e s y n t h e s i s (100). Nuclear methylase I I I l e v e l s a l s o r i s e c o r -respondingly i n G 2 (110). I t i s p o s s i b l e then t h a t h i s t o n e meth y l a t i o n has some r o l e i n subsequent chromatin condensation or m i t o s i s . The system i n which we chose to examine the process of h i s t o n e methylation was spermatogenesis i n the developing t r o u t t e s t i s . IV. Spermatogenesis In the immature t e s t i s , spermatogonial stem c e l l s -go through a s e r i e s of mitoses to produce primary spermatocytes. These c e l l s , upon two f u r t h e r m e i o t i c d i v i s i o n s , g i v e r i s e t o h a p l o i d spermatids, which then d i f f e r e n t i a t e f u r t h e r (spermio-genesis) t o g i v e mature sperm. Whereas spermatogenesis i n mammals i s a continuous process w i t h a l l c e l l types present i n the t e s t i s a t a given time (112), the same process i n Salmonids occurs once y e a r l y w i t h the r e l a t i v e l y synchronous appearance of c e l l types and i n c r e a s e i n t e s t i s weight (111). As i s the case f o r v e r t e b r a t e s (112), spermatogenesis i n Salmonid f i s h i s probably under the c o n t r o l of p i t u i t a r y -16-gonadotrophins (113). Hence, the process can be induced i n sexually immature trout (Salmo gal r drier i i ) by i n j e c t i o n s with salmon p i t u i t a r y extract (114,115); when so induced, the process i s more rapid and synchronous than normal. Following the early stages of spermatogenesis (up to primary spermatocyte), spermiogenesis i s accompanied by changes in c e l l morphology — chromatin condensation, development of propulsive t a i l , loss of cytoplasm and resultant decrease i n c e l l volume. These changes i n c e l l volume allow separation of d i f f e r e n t c e l l types on bovine serum albumin gradients (116), a technique used by Lam et a l . (117) to characterize the stages of mouse spermatogenesis. Louie and Dixon (93,94) used t h i s technique to l o c a l i z e , to s p e c i f i c c e l l types, the biochemical changes occurring during spermatogenesis. Thus, most of DNA, RNA and histone synthesis occurs i n the large d i p l o i d stem c e l l s and primary spermatocytes. These processes have stopped by the spermatid stage, accompanied by a decrease i n c e l l RNA content and subsequent replacement of histones by protamines. During t h i s replacement the protamines under-go an obligatory phosphorylation-dephosphorylation (92), necessary perhaps f o r binding to DNA and subsequent condensa-t i o n of chromatin. As noted e a r l i e r i n t h i s introduction, histone phosphorylation and acet y l a t i o n are associated mainly with the large d i p l o i d stem c e l l s and spermatocytes (78,93,94), while i n spermatids no phosphorylation, but some acet y l a t i o n occurs, perhaps as a necessary prelude to the replacement of -17-h i s t o n e s by protamine. S i n c e the s i t e s , k i n e t i c s and c e l l u l a r l o c a l i z a t i o n s o f h i s t o n e a c e t y l a t i o n and p h o s p h o r y l a t i o n are w e l l c h a r a c t e r i z e d i n t r o u t t e s t i s , we were i n t e r e s t e d i n l o o k i n g a t the r o l e o f ^ h i s t o n e m e t h y l a t i o n i n spermatogenesis. The l a r g e q u a n t i t i e s o f t i s s u e a v a i l a b l e , the easy p r e p a r a t i o n o f chromatin, the synchrony and ready s e p a r a t i o n of the d i f f e r e n t c e l l t y p e s , and the a b i l i t y o f c e l l suspensions t o i n c o r p o r a t e r a d i o a c t i v e p r e c u r s o r s — a l l a r e f u r t h e r good reasons f o r u s i n g t h i s system. P a r t A o f t h i s t h e s i s r e p o r t s on the s i t e s and some k i n e t i c s o f h i s t o n e m e t h y l a t i o n i n d e v e l o p i n g t r o u t t e s t i s . The r e s u l t s i n d i c a t e t h a t s p e c i f i c l y s y l r e s i d u e s of h i s t o n e s H3 and H4 are methylated, i n p o s i t i o n s homologous t o those determined f o r o t h e r organisms. T h i s m e t h y l a t i o n o c c u r s a s s o c i a t e d w i t h DNA and h i s t o n e s y n t h e s i s i n the l a r g e d i -p l o i d c e l l s . T h i s p r o c e s s i s p r o b a b l y not a s s o c i a t e d w i t h spermatids and the replacement o f h i s t o n e s by protamines. There i s n e g l i g i b l e t u r n o v e r of h i s t o n e methyl groups i n t r o u t t e s t i s c e l l s . The k i n e t i c s o f methyl l a b e l l i n g o f h i s t o n e H4 i n d i c a t e t h a t m e t h y l a t i o n i s a l a t e event i n the c e l l c y c l e a f t e r H4 s y n t h e s i s . -18-MATERIALS AND METHODS I . Chemicals and Abbreviations (a) Chemicals A l l chemicals obtained commercially were of the highest pur i t y or reagent grade. Sequencer Grade chemicals (Beckman Instruments Inc. or Pierce Chemical Co.) were used for pro-t e i n sequence studies with the Beckman 890 Sequencer. Radioactive compounds: - L-[methyl- 1 4C]methionine (56 mCi/mmole), L-[methyl- 3H]methionine (5 Ci/mmole), L-[ 1^C] lysi n e (340 mCi/mmole), DL-[4.5- 3H]lysine (20 Ci/mmole) and L-[5- 3H]arginine (8.9 Ci/mmole) - were obtained from Amersham-Searle Corp. Starch for gel electrophoresis was obtained from Connaught Laboratories and Ele c t r o s t a r c h Company. (b) Abbreviations dansyl c h l o r i d e : l,dimethylaminonaphthalene-5-sulfonyl chloride POPOP: 1,4-Bis-(5-phenyloxazol-2-yl)-benzene PPO: 2,5-diphenyloxazole TCA-tungstate: 5% t r i c h l o r o a c e t i c acid, 0.25% sodium tungstate, pH 2 TMKS buffer: Tris-HCl (50 mM, pH 7.4), MgClz (1 mM), KC1 (25 mM) and sucrose (0.25 M) monomethyl-lysine: e-N-monomethyl-L-lysine N -methyl-histidme: 3-methyl-histxdine -19-dimethyl-lysine: e-N-dimethyl-L-lysine t r i m e t h y l - l y s i n e : £-N-trimethyl-L-lysine I I . I s o l a t i o n and Characterization of iri vivo [ 1 ''C]methyl-ated Trout Tes t i s Histones (a) [ 1 **C]methyl l a b e l l i n g of histones Testes were obtained from rainbow trout i n which sperm-atogenesis had been induced by twice-weekly i n j e c t i o n of salmon p i t u i t a r y extracts (114). Testes were scissor-minced i n 2 volumes of TMKS buffer containing 0.1% glucose, and were then gently homogenized i n a Potter-Elvehjem homogenizer. The r e s u l t i n g c e l l suspension was f i l t e r e d through cheese-c l o t h , and was then incubated with 20 yCi/ml of L-[methyl-1''C] methionine at 16-18°C for up to 7 hours i n the presence of 100 U/ml of p e n i c i l l i n and streptomycin (Baltimore Bio-l o g i c a l ) , and supplemented with one tenth volume of T r i s -buffered Waymouth's medium (118) as described by Louie and Dixon ( 84). C e l l s were c o l l e c t e d by ce n t r i f u g a t i o n , homo-genized vigorously i n TMKS buffer, and n u c l e i were p e l l e t e d at 1,000 g for 10 min. Af t e r two more washings of n u c l e i with TMKS, chromatin was prepared by washing nu c l e i further with 0.15 M NaCl, 20 mM EDTA and then three times i n 10 mM T r i s pH 8.0 ( 9 ). The histones were extracted from the viscous chromatin p e l l e t with 0.4 N H 2 SOi t and p r e c i p i t a t e d with 4 volumes of ethanol at -20°C. The histones were then -20-d i s s o l v e d i n water, loaded on a c a r b o x y m e t h y l - c e l l u l o s e column (Bio-Rad L a b o r a t o r i e s ) and h i s t o n e s HI and H6 were p u r i f i e d by L i C l g r a d i e n t e l u t i o n ( 9 ). A f t e r the removal of h i s -tones H6 and H i , the other h i s t o n e s were e l u t e d from the column w i t h 0.2 N HCl, concentrated by l y o p h i l i z a t i o n , and separated on long Bio-Gel P-10 (Bio-Rad L a b o r a t o r i e s ) columns i n 0.01 N HCl ( 40). When r e q u i r e d , protamine was separated from the h i s t o n e s on a s h o r t e r (2 x 100 cm) P-10 column, p r i o r t o t h i s step. For r a d i o a c t i v i t y a n a l y s i s , a l i q u o t s of h i s t o n e were counted i n Bray's s o l u t i o n (119), w h i l e pro-t e i n c o n c e n t r a t i o n was monitored by absorbance a t 230 nm. (b) S t a r c h g e l e l e c t r o p h o r e s i s Histone p u r i t y was monitored by e l e c t r o p h o r e s i s on urea-aluminum l a c t a t e s t a r c h g e l s , prepared as d e s c r i b e d by Sung and Smithies (120). Histone samples were elect r o p h o r e s e d at 6 volts/cm, 30 m i l l i a m p e r e s w i t h water-cooled g e l t r a y s at 4°C. Gels were h o r i z o n t a l l y t r i s e c t e d , and middle or bottom s l i c e s were s t a i n e d 40 min w i t h 0.1% Amido Black con-t a i n i n g C o C l 2 , and destained i n N H 2 S O 4 (120). (c) Determination of methylated amino a c i d s In p r e l i m i n a r y analyses, [ 1 ''C]methyl-labelled p r o t e i n was hydrolyzed i n 6 N HCl i n vacuo, a t 110°C f o r 22 hours. Hydrolysates were electrophoresed on Whatman 3 MM paper a t -21-pH 6.5 ( p y r i d i n e / a c e t i c a c i d /water, 100:4 :900) w i t h methio-nine and v a r i o u s m e t h y l - l y s i n e standards as f o l l o w s : mono-m e t h y l - l y s i n e (Sigma), d i m e t h y l - l y s i n e (Cyclo C h e m i c a l ) , and t r i m e t h y l - l y s i n e (a generous g i f t from Dr. Robert DeLange). L a b e l l e d amino a c i d s were l o c a t e d by autora d i o g r a p h y on Kodak Bl u e Brand X-ray f i l m and e l u t e d w i t h 25% a c e t i c a c i d . R a d i o a c t i v i t y was determined on a l i q u o t s i n Bray's s o l u t i o n . L a t e r , a c i d h y d r o l y s a t e s were chromatographed on a Technicon amino a c i d a n a l y z e r w i t h Durrum DC1A r e s i n (Durrum Instruments) a t 41°C i n 0.43 M c i t r a t e , pH 6.1. To d e t e r -mine r a d i o a c t i v i t y i n the methyl amino a c i d s , the column e f f l u e n t was d i v e r t e d from the r e a c t i o n c o i l and 1.0 ml f r a c t i o n s were c o l l e c t e d . R a d i o a c t i v i t y was determined on an a l i q u o t d i s s o l v e d i n Aquasol (New England N u c l e a r ) . (d) D e t e r m i n a t i o n o f c e l l u l a r L-methionine and S-adenosyl-L-methionine T e s t i s c e l l s were i n c u b a t e d i n TMKS - 0.1% g l u c o s e w i t h L - [ m e t h y l - 1 ^ C ] m e t h i o n i n e f o r 10-30 min, then i n t r a c e l l u l a r . [ 1 "*C] methionine and [ 1 **C] S-adenosyl-L-methionine were d e t e r -mined by the method o f S a l v a t o r e e t a l . (121). In t h i s p r o -cedure, p e r c h l o r i c a c i d e x t r a c t s o f c o l l e c t e d c e l l s were a p p l i e d t o Dowex-1 (X8) (Bio-Rad) columns and e l u t e d w i t h 2 N H2SOi» ( f o r methionine) and 6 N H 2 S O 4 ( f o r S-adenosyl-L-methionine) . -22-I I I . Sites of i n vivo Methylation i n Trout T e s t i s Histones (a) Automated protein sequencing Samples of [^C]methyl l a b e l l e d histone, 0.5-1.0 ymoles i n 0.6 ml of N a c e t i c acid, were subjected to Edman degrada-tions i n a Beckman 890 protein sequencer, using v o l a t i l e reagents as described by N i a l l et a l . (122). In t h i s DMAA program, phenylisothiocyanate buffered with dimethylallylamine (DMAA) t r i f l u o r o a c e t i c acid i s reacted with protein, followed by a benzene wash, double cleavage with heptafluorobutyric acid, and extraction of the thiazolinone d e r i v a t i v e with butyl c h l o r i d e . The 2-anilino-5-thiazolinone d e r i v a t i v e s of the amino acids were then converted to the 3-phenyl-2-thiohydantoin (PTH) derivatives by incubation i n N HCl at 80°C for 10 min under N2 (123). Amino acids were then i d e n t i f i e d on a Beckman 120C or Technicon amino acid analyser a f t e r hydrolysis of the PTH derivatives i n 6 N HCl at 140°C for 24 hr (124). Radioactivity was determined on aliquots of the 2-anilino-5-thiazolinone derivatives dried down and redissolved i n Bray's so l u t i o n , as described by Candido and Dixon ( 22) „ (b) [1UC]methyl peptides of histone H3 50 mg of histone H3 (180,000 cpm) was reacted with 1 ml aliquots of N-bromosuccinimide (6 mg/ml i n 50% a c e t i c acid) over 2 hours at 20°C u n t i l there was no further increase i n -23-A2 6 0. A f t e r l y o p h i l i z a t i o n , the r e s u l t i n g p e p t i d e s were sep-a r a t e d on a Sephadex G-50 column (Pharmacia), 2 x 180 cm, i n 25% a c e t i c a c i d . The r a d i o a c t i v e peak, c o n c e n t r a t e d by l y o p h i l i z a t i o n , was-then rechromatographed on the same c o l -umn. P r o t e i n was monitored by r e a c t i o n w i t h n i n h y d r i n and measurement o f subsequent absorbance a t 570 nm. The s i n g l e r a d i o a c t i v e p e p t i d e so i s o l a t e d , of amino a c i d c o m p o s i t i o n c o r r e s p o n d i n g t o the N - t e r m i n a l 40 amino a c i d s of h i s t o n e H3 (17-21), was next m a l e y l a t e d w i t h 30 mg of m a l e i c anhydride i n 0.1 M NaHCG-3, pH 8.5, e s s e n t i a l l y as d e s c r i b e d by B u t l e r e t a l . (125). The maley-l a t e d p e p t i d e was then d i g e s t e d w i t h t r y p s i n (1:50 by weight) i n 0.1 M ammonium b i c a r b o n a t e and demaleylated i n 0.05 M p y r i d i n e - a c e t a t e pH 3.2 f o r 40 hours a t 37°C. The r e s u l t i n g p e p t i d e s were chromatographed on a Sephadex G-10 (Pharmacia) column, 2.5 x 200 cm, i n 10% a c e t i c a c i d . R a d i o a c t i v e f r a c -t i o n s were p o o l e d , c o n c e n t r a t e d and chromatographed on Whatman 3 MM paper i n p y r i d i n e : b u t a n o l : a c e t i c a c i d s w a t e r , 15:10:3:12 ( 12,126). The r a d i o a c t i v e p e p t i d e s , l o c a t e d by autoradiography w i t h Kodak Blue Brand X-ray f i l m , were c u t out and e l u t e d w i t h 25% a c e t i c a c i d . (c) T r y p t i c p e p t i d e s o f [ l l fC]methyl l a b e l l e d h i s t o n e H4 70 mg of h i s t o n e H4 (150,000 cpm) was m a l e y l a t e d w i t h 150 mg of m a l e i c anhydride (125), d i g e s t e d w i t h t r y p s i n -24-(1:50) and demaleylated i n p y r i d i n e - a c e t a t e pH 3.2 as des-c r i b e d above f o r h i s t o n e H3. The r e s u l t i n g p e p t i d e s were chromatographed on a Sephadex G-25 column i n 25% a c e t i c a c i d , and the r a d i o a c t i v e peak was c o n c e n t r a t e d and chromatographed on Whatman 3 MM paper as d e s c r i b e d above f o r h i s t o n e H3. The s i n g l e r a d i o a c t i v e p e p t i d e was c u t out and e l u t e d w i t h 25% a c e t i c a c i d . (d) Sequence d e t e r m i n a t i o n s on [ ^ C ] m e t h y l l a b e l l e d  t r y p t i c p e p t i d e s A m o d i f i c a t i o n o f the Edman d e g r a d a t i o n (127) was pe r -formed on 0.1 t o 0.2 ymoles o f p e p t i d e , as d e s c r i b e d by Candido and Dixon ( 40 ). B r i e f l y , the procedure i n v o l v e s c o u p l i n g w i t h p h e n y l i s o t h i o c y a n a t e i n p y r i d i n e + N - e t h y l -morpholine b u f f e r , d r y i n g under vacuum, c y c l i z a t i o n i n anhydrous t r i f l u o r o a c e t i c a c i d , d r y i n g , and e t h y l a c e t a t e e x t r a c t i o n o f the t h i a z o l i n o n e d e r i v a t i v e o f the amino a c i d . B e f o r e each Edman s t e p , an a l i q u o t o f p e p t i d e was removed f o r amino a c i d a n a l y s i s ( s u b t r a c t i v e Edman sequencing) o r d a n s y l a t i o n a c c o r d i n g t o Gray (128) as m o d i f i e d by H a r t l e y (129). A l i q u o t s o f the r e l e a s e d 2 - a n i l i n o - 5 - t h i a z o l i n o n e d e r i v a t i v e s were an a l y z e d f o r r a d i o a c t i v i t y by d r y i n g and r e d i s s o l v i n g i n Bray's s o l u t i o n . The d a n s y l a t i o n procedure i n v o l v e d r e a c t i n g the p e p t i d e a l i q u o t i n 0.1 M NaHC03 w i t h d a n s y l c h l o r i d e (2.5 mg/ml) i n an equal volume of acetone a t 37°C f o r 2 hours, then -25-drying i n vacuo. The dansyl d e r i v a t i v e of the N H 2-terminal amino acid was l i b e r a t e d by hydrolysis (in 6 N HC1 at 110°C for 16 hr) and was i d e n t i f i e d by two-dimensional chromato-graphy (129) on 5 x 5 cm polyamide sheets (Cheng Chin Trad-ing Co., T a i p e i ; d i s t r i b u t e d by Gallard-Schlesinger, Carle Place, N.Y.). Solvent systems used were as follows: 1st dimension - Solvent 1, 1.5% formic acid 2nd dimension - Solvent 2, benzene:acetic acid, 9:1 Solvent 3, ethyl acetate:methanol:acetic acid, 20:1:1 IV. Histone Methylation i n D i f f e r e n t C e l l Types from Develop-ing Trout T e s t i s (a) C e l l separations T e s t i s c e l l suspensions were prepared from hormonally-induced t e s t i s (as i n Materials & Methods, section II (a)) and aliquots of 0.5 ml (1 to 7 x 10 8 c e l l s ) were then incu-bated with 5 yCi/ml of L - [ 1 ^ C ] l y s i n e and 2 mCi/ml of L-[methyl- 3H]methionine at 16°C f o r 2 hours. C e l l s were c o l -lected by centrifugation, resuspended i n phosphate-buffered saline (0.9% NaCl) and a 15 ml aliq u o t containing 7 to 9 x 10 7 c e l l s was allowed to sediment at unit gravity on a 600 ml, 1-3% bovine serum albumin gradient f o r 255 min at 4°C ( 93 / 117). Then 8 5 f r a c t i o n s of 7 ml each were c o l l e c t e d from the gradient i n 45 minutes, so that the t o t a l c e l l separation -26-time was 300 minutes (5 hours), as d e t a i l e d by Louie and Dixon ( 93). C e l l s were counted on a hemocytometer. (b) Starch gel electrophoresis for histones C e l l s of d i f f e r e n t types, c o l l e c t e d from the gradient, were f i l t e r e d onto glass f i b e r f i l t e r s ( M i l l i p o r e Corp., AP 20-025-00), washed with phosphate-buffered s a l i n e , and fix e d with ethanol. The f i l t e r s were then extracted d i r e c t l y i n 0.4 N HCl i n the s l o t s of a urea-lactate starch gel and electrophoresed, as described by Louie and Dixon ( 9 3 ) . Gels were h o r i z o n t a l l y t r i s e c t e d ; the bottom slab was stained with cobalt-Amido Black (Schwartz-Mann) for histones (120), while the middle slab was used for analysis of radio-a c t i v i t y i n the histones, as described below. (c) Pulse-chase turnover experiments 0.5 ml of a t e s t i s c e l l suspension (5 x 10 8 c e l l s ) was incubated with 100 yCi/ml of DL-[4,5- 3H]lysine and 50 uCi/ml L-[methyl- 1^C]methionine for 2 hours at 16°C as described above. C e l l s were then c o l l e c t e d by cent r i f u g a t i o n and re-suspended i n 8 ml of TMKS - 0.1% glucose with streptomycin, p e n i c i l l i n , T r is-buffered Way-mouth* s medium (see Materials and Methods, section I I (a)), and supplemented with 1 mM L-lysine and L-methionine to d i l u t e radioactive precursors present. During further incubation at 16°C, 1 ml aliquots -27-(5 x 1 0 7 c e l l s ) were f i l t e r e d onto g l a s s f i b e r f i l t e r s , c e l l s were f i x e d and r a d i o a c t i v i t y i n the hi s t o n e s was d e t e r -mined on s t a r c h g e l s as de s c r i b e d below. C e l l v i a b i l i t y d u r i n g t h i s f u r t h e r "chase" i n c u b a t i o n was monitored as f o l l o w s . A f t e r the i n i t i a l 2 hr i n c u b a t i o n , c e n t r i f u g a t i o n and resuspension, a n a l i q u o t of the c e l l s was f u r t h e r incubated w i t h 50 yci/ml of L - [ 3 H ] a r g i n i n e and t o t a l r a d i o a c t i v i t y i n c o r p o r a t e d i n t o c e l l n u c l e i at v a r y i n g times was determined as described below. (d) R a d i o a c t i v i t y a n a l y s i s T o t a l r a d i o a c t i v i t y i n c o r p o r a t e d i n t o c e l l n u c l e i from c e l l s e p a r a t i o n g r a d i e n t f r a c t i o n s was determined by pre-c i p i t a t i o n w i t h TCA-tungstate on g l a s s f i b e r f i l t e r s ( 9 3 ) . R a d i o a c t i v i t y i n the h i s t o n e s was determined from the middle s l a b of the s t a r c h g e l , which was c u t i n t o 2 mm s l i c e s . These s l i c e s were incubated i n s c i n t i l l a t i o n v i a l s w i t h 0.5 ml NCS reagent (Amersham-Searle) o v e r n i g h t , then 5 ml of toluene s c i n t i l l a t i o n f l u i d (toluene + 0.01% POPOP and 0.4% PPO) was added and the v i a l s incubated a t 45°C f o r 3 hours p r i o r to c o o l i n g and counting ( 93 ) . V. K i n e t i c s of Methyl L a b e l l i n g of Histone H4 (a) P r e p a r a t i o n of [^C] methyl l a b e l l e d histone H4 A t e s t i s c e l l suspension was incubated w i t h 20 yCi/ml of L-[methyl- 1''C]methionine, supplemented w i t h p e n i c i l l i n , -28-streptomycin and Waymouth's medium minus methionine (Materials & Methods, section II (a)] and at varying time i n t e r v a l s up to 12 hr, aliquots were taken and histone H4 was prepared by a c i d extraction of chromatin, and chromatography on carboxymethylcellulose and Bio-Gel P-10 columns [Materials and Methods, section II (a)]. (b) Radioactivity i n the modified species of histone H4 Histone H4 samples, prepared a f t e r varying times of incubation, were electrophoresed on urea-lactate starch gels. Radioactivity i n the acetylated and phosphorylated H4 species was then determined, as described by Louie and Dixon ( 84), from starch gel s l i c e s [Materials and Methods, section IV (d)]. -29-RESULTS I. I s o l a t i o n and C h a r a c t e r i z a t i o n o f i n v i v o M e t h y l a t e d T r o u t T e s t i s H i s t o n e s At the time t h i s i n v e s t i g a t i o n was s t a r t e d , t h e r e were many r e p o r t s o f p o s t s y n t h e t i c (100) m e t h y l a t i o n o f h i s t o n e s -predominently H3 and H4 ( 12 , 17 , 21 ,100) , but a l s o H2A, H2B - from v a r i o u s s o u r c e s . Furthermore, a wide v a r i e t y o f methyl amino a c i d s - e-N-mono,di- and t r i m e t h y l - l y s i n e ( 12 , 21 ,100), as w e l l as N -methyl and d i m e t h y l - a r g i n i n e d e r i v a t i v e s (100) and N - m e t h y l - h i s t i d i n e (43) were found. In d e v e l o p i n g t r o u t t e s t i s , t h e r e i s a synchronous development o f c e l l s - spermatogonia spermatocytes -*• spermatids sperm - accompanied by s t r i k i n g m o r p h o l o g i c a l and b i o c h e m i c a l changes, i n c l u d i n g the l o s s o f h i s t o n e s and appearance o f the s p e r m - s p e c i f i c protamines. In t h i s model system f o r d i f f e r e n t i a t i o n , h i s t o n e a c e t y l a t i o n and phos-p h o r y l a t i o n had been e x t e n s i v e l y c h a r a c t e r i z e d by Dixon and coworkers ( 28 , 62) . We were t h e r e f o r e i n t e r e s t e d i n the nature and r o l e o f h i s t o n e m e t h y l a t i o n i n the development of t h i s t i s s u e . T h i s f i r s t s e c t i o n d e a l s w i t h i d e n t i f y i n g which t e s t i s h i s t o n e s are methylated, and which methyl amino a c i d s are p r e s e n t , f o l l o w i n g i n c u b a t i o n o f t e s t i s c e l l s w i t h L-[methyl-1 ''C] methionine. / -30-(a) Which histones are methylated Histones were prepared from such an incubation, as des-cribed i n Materials and Methods. Figure 1 shows the L i C l gradient e l u t i o n of histones H6 and Hi from a carboxymethyl-c e l l u l o s e column. Amino acid analyses of H6 and Hi show that neither histone contains methionine, yet H6 showed some [1''C] incorporation and so was possibly methylated. When protamine was present i n the sample, i t was sep-arated from histones on a Bio-Gel P-10 column (Fig. 2). The [lf*C] incorporation into protamine proved to be as N-terminal methionine (as judged by 99% loss of r a d i o a c t i v i t y a f t e r 1 Edman degradation), as described by Wigle and Dixon (130) . The histones, recovered from the carboxymethylcellulose column, were then fractionated on a long Bio-Gel P-10 column (Fig. 3). Histones H3 and H4 were the predominant l a b e l l e d species, with some perhaps i n H2B. Since histone H2B con-tains roughly the same proportion of i n t e r n a l methionine as H3 and H4, i t was s u r p r i s i n g that so much more [^C] l a b e l appeared i n H3 and H4. (b) Which methylated basic amino acids are present The nature of the [^C] l a b e l incorporated into each of the histones was next examined, by acid hydrolysis of the histones and preliminary f r a c t i o n a t i o n of amino acids by pH 6.5 electrophoresis. The methyl basic amino acids are stable -31-FIG. 1. L i C l g r a d i e n t s e p a r a t i o n o f [ 1 1 1C]methyl l a b e l l e d h i s -tones. T e s t i s c e l l s were i n c u b a t e d w i t h [methyl- 1 "*C] methionine and the h i s t o n e s were prepared and f r a c t i o n a t e d on a 2 x 20 cm c a r b o x y m e t h y l - c e l l u l o s e column w i t h a 400 ml 0.15 to 0.75 M L i C l g r a d i e n t . P r o t e i n was monitored by f o l l o w i n g the A 2 3 0 . F o l l o w i n g the e l u t i o n o f H6, the g r a d i e n t was stopped and the r e s t o f the h i s t o n e s were washed from the column w i t h 0.2 N HCl. -32-FIG. 2. The s e p a r a t i o n of [ ^ C ] m e t h y l l a b e l l e d h i s t o n e s from protamines. 1 ' ' C - l a b e l l e d h i s t o n e s and protamines were chrom-atographed on a 2 x 100 cm B i o - G e l P-10 column i n 0.01 N HCl. P r o t e i n was monitored b y A 2 3 0• -33-2000 H1200 400 c r .0 : C « (TJ w O _ 8 5 •5 o fl) Q. 5 o 80 120 160 FRACTION NO., 6ml. 200 FIG. 3. P r e p a r a t i o n of [^C]methyl l a b e l l e d h i s t o n e f r a c t i o n s on Bxo-Gel P-10. The histones (minus HI, H6), recovered from Bio-Gel P-10 chromatography ( F i g . 2), were chromatographed on a long (3 x 360 cm) Bio-Gel P-10 column i n 0.01 N HC1. P r o t e i n was monitored by A 2 3 0. -34-to a c i d h y d r o l y s i s (100) , and move r a p i d l y towards the cathode, whereas methionine i s l e s s h i g h l y charged and s t a y s near the o r i g i n ( F i g . 4 ) . I t was e v i d e n t ( F i g . 4) t h a t h i s t o n e s HI, H2A and protamine c o n t a i n e d no methylated b a s i c amino a c i d s , whereas H6, H2B, H3 and H4 d i d . The p r o p o r t i o n o f [ 1 "*C]methyl amino a c i d t o [ 1 ^ C] i n t e r n a l methionine i s s u r p r i s i n g l y h i g h (90%) and accounts f o r the h i g h i n c o r p o r a t i o n i n t o H3 and H4 r e l a t i v e to H2B. One e x p l a n a t i o n o f t h i s r e s u l t i s t h a t most o f the [methyl- 1^C]methionine i n the c e l l s i s converted t o the methyl donor S-adenosyl [methyl- 1^C]methionine (SAM). Examination of the l e v e l s of methionine and SAM by the method of S a l v a t o r e et a l . (121) show t h a t t h i s may indeed be the case, as g r e a t e r than 80% o f the c e l l r a d i o a c t i v i t y i s r e c o v e r a b l e as SAM. T h i s r e s u l t o b v i a t e d the need to use cycl o h e x i m i d e t o i n h i b i t p r o t e i n s y n t h e s i s and thus minimize i n t e r n a l [1'*C] methionine i n c o r p o r a t i o n . Such treatment would have lowered c e l l v i a b i l i t y over the long i n c u b a t i o n times used f o r some experiments. • . • . To i d e n t i f y which methyl amino a c i d s were p r e s e n t , a c i d h y d r o l y s a t e s of h i s t o n e s were chromatographed on a Technicon amino a c i d a n a l y z e r as d e s c r i b e d i n M a t e r i a l s and Methods. A standard run i s shown i n F i g . 5. H y d r o l y s a t e s o f whole, h i s t o n e were found t o c o n t a i n [ l l fC] mono-, d i - and t r i m e t h y l -l y s i n e , but no r a d i o a c t i v i t y o r n i n h y d r i n p o s i t i v e m a t e r i a l -35-FIG. 4. The presence of [ 1 ''C]methionine and [ l l 4C]methyl amino acids i n the histones. Acid hydrolysates of the histones (6N HCl, 20 hr) were electrophoresed at pH 6.5 on Whatman 3 Mil paper as described i n the text. 1 4 C - r a d i o a c t i v i t y was located by autoradiography on Kodak Blue Brand X-ray f i l m . [ 1 !*C]methyl amino acids moved rapidl y with methyl green marker towards the cathode (spots at the top of the chromatogram), whereas [ I l +C]-methionine remained near the o r i g i n . -36-^ 0.2 240 270 300 330 T I M E , m i n FIG. 5. S e p a r a t i o n o f e-N-methyl-lysines (mono, d i - and t r i -methyl d e r i v a t i v e s ) on a Technicon amino a c i d a n a l y s e r . Methyl-amino a c i d standards were chromatographed on Durrum DC1A r e s i n (Durrum Instruments) a t 41°C i n 0.43 M c i t r a t e , pH 6.1. A flow r a t e of 12.5 ml/hr was used f o r the 55 x 0.9 cm a n a l y s e r column. The e l u t i o n p o s i t i o n s of h i s t i d i n e (His) and N T-methyl-h i s t i d i n e [His(xMe)] are marked by arrows. Ammonia, a r g i n i n e and i t s d e r i v a t i v e s e l u t e l a t e r from the column. -37-was found i n the N - m e t h y l - h i s t i d i n e o r m e t h y l - a r g i n i n e por-t i o n s o f the chromatogram. H i s t o n e H3 c o n t a i n e d mono-, d i - and t r i m e t h y l - l y s i n e i n the r a t i o 0.49:0.61:0.18 r e s i d u e s per molecule. H i s t o n e H4 c o n t a i n e d o n l y mono- ar.d d i m e t h y l - l y s i n e , 0.43:0.27 r e s i -dues. D e s p i t e the f i n d i n g , a f t e r pH 6.5 e l e c t r o p h o r e s i s , of s m a l l amounts o f [ 1 ^ C] b a s i c amino a c i d i n h i s t o n e H6, no n i n h y d r i n - p o s i t i v e m a t e r i a l c o u l d be d e t e c t e d from H6 h y d r o l y s a t e s i n the m e t h y l - l y s i n e r e g i o n s o f the chromato-gram. T h i s might be due t o r a d i o a c t i v e l a b e l l i n g o f o n l y t r a c e amounts o f H6, as was r e p o r t e d f o r N - m e t h y l - h i s t i d i n e i n H i and H5 ( 43 ) from b i r d e r y t h r o c y t e s . A l t e r n a t i v e l y , the [ll*C] l a b e l i n H6 c o u l d r e p r e s e n t low l e v e l s o f con-t a m i n a t i o n by another [ 1 ^ C ] m e t h y l a t e d molecule (e.g. H3, H4). S i m i l a r l y H2B showed n e g l i g i b l e amounts o f m e t h y l - l y s i n e s upon a n a l y s i s . Here, c o n t a m i n a t i o n w i t h s m a l l amounts o f h i s t o n e H3 was v e r y l i k e l y (see F i g . 3 ) . S i n c e H3, H4 and p o s s i b l y H6, and H2B c o n t a i n e d methyl-l y s i n e s , they were examined f u r t h e r t o l o c a l i z e which l y s y l r e s i d u e s i n the p r o t e i n were methylated. I . S i t e s of i n v i v o M e t h y l a t i o n o f L y s y l Residues i n T e s t i s H i s t o n e s (.a) H i s t o n e H3 The NH2-terminal o f t h i s h i s t o n e i s not bl o c k e d , so t h a t -38-i t was f e a s i b l e to perform automated Edman d e g r a d a t i o n s on t ^ C ] m e t h y l - l a b e l l e d h i s t o n e I I I . As m e t h y l - l y s i n e s are chem-i c a l l y s t a b l e to the sequencing procedures, the p o s i t i o n s of t ^ C ] r a d i o a c t i v i t y and hence o f methyl groups c o u l d be l o c a l i z e d . T h i s t echnique was s u c c e s s f u l l y a p p l i e d by Candido and Dixon (22 ) who sequenced the f i r s t 25 r e s i d u e s of t e s t i s h i s t o n e H3, and l o c a l i z e d [ 1 "*C] a c e t a t e a t l y s y l r e s i d u e s 9, 14, 18 and 23. In F i g . 6, the r a d i o a c t i v i t y r e c o v e r e d i n the c h l o r o -butane f r a c t i o n s from the sequenator i s p l o t t e d a g a i n s t the number o f d e g r a d a t i o n s performed on [ 1^C]methyl l a b e l l e d H3. F o r each v a l u e , the background r a d i o a c t i v i t y was s u b t r a c t e d , then v a l u e s were c o r r e c t e d f o r a r e p e t i t i v e y i e l d o f 93%, determined from a s e m i - l o g a r i t h m i c p l o t o f y i e l d o f a l a n y l r e s i d u e s vs number o f d e g r a d a t i o n s ( F i g . 7) as d e s c r i b e d by Candido and Dixon (22 ) . There a r e two major peaks a t l y s i n e s 9 and 27, and one minor peak a t l y s i n e 4, i n d i c a t i n g a t l e a s t t h r e e s i t e s o f e-N-methylation o f l y s i n e s i n h i s t o n e H3. A s m a l l r e p r o -d u c i b l e peak a t l y s i n e 36, of 1,000 cpm (4,000 cpm when c o r r e c t e d f o r r e p e t i t i v e y i e l d ) above a background o f 1,200 cpm was a l s o d e t e c t e d , but a 93% r e p e t i t i v e y i e l d p r e c l u d e s good r e s o l u t i o n a f t e r t h i s number o f d e g r a d a t i o n s . The r e c o v e r y o f [^C] r a d i o a c t i v i t y was not complete -39-T NO. OF DEGRADATIONS FIG. 6. Automated d e g r a d a t i o n of [ 1 4 C ] m e t h y l l a b e l l e d t e s t i s h i s t o n e H3. 12 mg o f h i s t o n e H3 (500,000 cpm) was sequenced i n a Beckman 890 p r o t e i n sequencer, u s i n g v o l a t i l e reagents (122). R a d i o a c t i v i t y r e c o v e r e d a t each d e g r a d a t i v e step has been c o r r e c t e d f o r a r e p e t i t i v e y i e l d o f 93%, based on r e c o v e r y of a l a n y l r e s i d u e s , Y = 1 l o g B, where Y i s the r e -N - 1 p e t i t i v e y i e l d , N i s the number of deg r a d a t i o n s and B i s the rec o v e r y o f amino a c i d a t s t e p N. So f o r Y = 93%, a t r e s i d u e 9 l o g (0.93) = 1 l o g B and B = 0.55. The r a d i o a c t i v i t y 9 - 1 at s t e p 9 i s t h e r e f o r e c o r r e c t e d by a f a c t o r o f 1 . 0.55 -40-FIG. 7. Recovery o f a l a n i n e d u r i n g automated s e q u e n t i a l deg-r a d a t i o n o f h i s t o n e H 3 . The l o g a r i t h m o f the r e l a t i v e y i e l d of a l a n i n e , p r e s e n t i n the sequence a t r e s i d u e s 1,7,15,21,24,25, i s p l o t t e d a g a i n s t number of d e g r a d a t i o n performed. The N H 2 -t e r m i n a l a l a n i n e i s taken as 100% r e l a t i v e y i e l d . S i n c e Log Y = T r t — r - M ~ 1 B / A Y = r e p e t i t i v e y i e l d ^ B ~ A A, B are y i e l d s o f r e s i d u e s a f t e r N, o r N„ d e g r a d a t i o n s A B ^ the s l o p e o f the above p l o t , l o g B/A vs N n - N a, g i v e s l o g Y. -41-from the sequencer. To c o n f i r m t h a t m e t h y l a t i o n o f H3 i s con-f i n e d t o the N-terminus o f the molecule, 50 mg of H3 w i t h 180,000 cpm of i n c o r p o r a t e d [ l l fC] methyl groups, was c l e a v e d w i t h N-bromo-succinimide i n 50% a c e t i c a c i d . When the r e s u l t i n g p e p t i d e s were chromatographed on Sephadex G-50, almost a l l [ 1 ^ C ] i n c o r p o r a t i o n was i n a s i n g l e r a d i o a c t i v e p e p t i d e NBS-1 (F i g u r e 8) which, when rechrom-atographed on Sephadex G-50 or c a r b o x y m e t h y l c e l l u l o s e had an amino a c i d c o m p o s i t i o n c o r r e s p o n d i n g t o the N - t e r m i n a l 40 amino a c i d s expected from an N-bromosuccinimide cleav a g e at r e s i d u e 41, which i s a t y r o s i n e i n the h i s t o n e H3 o f v a r i o u s s p e c i e s (17-21). The r e s t o f the r a d i o a c t i v i t y c o u l d be accounted f o r as u n d i g e s t e d m a t e r i a l ( F i g . 8, f r a c t i o n s 20-30) or low l e v e l s o f i n c o r p o r a t i o n i n t o i n t e r n a l methionine ( F i g . 8, f r a c t i o n s on e i t h e r s i d e o f the peak). The m e t h y l a t i o n s i t e s determined by automatic sequencing were then confirmed by i s o l a t i n g l a b e l l e d t r y p t i c p e p t i d e s of t h i s N - t e r m i n a l p e p t i d e , NBS-1. F o l l o w i n g m a l e y l a t i o n , t r y p s i n d i g e s t i o n , d e m a l e y l a t i o n and chromatography on Seph-adex G-10 ( F i g . 9) and on Whatman 3 MM paper, t h r e e major r a d i o a c t i v e p e p t i d e s B l , B2 and B3 were i s o l a t e d (Table I I I ) . P e p t i d e B3 corresponds t o r e s i d u e s 27-40 f o r h i s t o n e H3 and i s approximately h a l f - m e t h y l a t e d a t r e s i d u e 27, predom-FIG. 8. Sephadex G-50 chromatography of N-bromosuccinimide de-r i v e d p e p t i d e s of [^C]methyl l a b e l l e d h i s t o n e H3. 50 mg of h i s t o n e H3 was r e a c t e d w i t h N-bromosuccinimide i n 50% a c e t i c a c i d , and the r e s u l t i n g p e p t i d e s were l y o p h i l i z e d and separated on a Sephadex G-50 column i n 25% a c e t i c a c i d . P r o t e i n concen-t r a t i o n was monitored by r e a c t i o n w i t h n i n h y d r i n and measure-ment o f the absorbance a t 570 nm. -43-20 40 60 Fraction No., 2 m l FIG. 9. Sephadex G-10 chromatography of H3 t r y p t i c p e p t i d e s . [^C] methyl l a b e l l e d p e p t i d e NBS-1, d e r i v e d from H3 by N-bromo-su c c i n i m i d e d i g e s t i o n , was m a l e y l a t e d and d i g e s t e d w i t h t r y p s i n . The r e s u l t i n g p e p t i d e s were f r a c t i o n a t e d on a 1.5 x 90 cm Sephadex G-10 column, i n 10% a c e t i c a c i d . P e p t i d e was monitored by the n i n h y d r i n r e a c t i o n a t 570 nm. -44-TABLE I I I [^C] methyl l a b e l l e d peptides derived from histone H3 P o s i t i o n i n Peptide N-terminal residue Histone H3 Sequence 3 residues # e-N -methyl-lysines per peptide mono- d i - t r i -NBS-l b alanine 1-40 0.49 0.61 0.18 B - l c l y s i n e 9-17 0.09 0.13 0.24 B-2 C l y s i n e 9-17 0.29 0.45 0.06 B-3 C l y s i n e 27-4 0 0.22 0.24 0.02 Peptides were placed by amino acid composition, N-terminal analysis and homology with the histone H3 of other species (10,11,13,15,16). Derived from histone H3 by digestion with N-bromosuccinimide. Only h a l f (75,000 cpm) of the peptide was maleylated and t r y p s i n digested. Derived by t r y p t i c digestion of maleylated NBS-1. i n a n t l y as equal p r o p o r t i o n s o f the mono- and d i m e t h y l - l y s i n e d e r i v a t i v e s (Table I I I ) . P e p t i d e s B l and B2, r e c o v e r e d i n approximately equal amounts, both correspond t o r e s i d u e s 9-17 i n h i s t o n e H3 (Table I I I ) . B l , however, has a lower m o b i l i t y than B2 on paper chromatography i n b u t a n o l : p y r i d i n e : a c e t i c acid:water o r upon e l e c t r o p h o r e s i s a t pH 6.5, s u g g e s t i n g t h a t B l has one l e s s p o s i t i v e charge than B2 (131). B l pro b a b l y d i f f e r s from B2 then, i n b e i n g a c e t y l a t e d a t l y s i n e 14 ( 15 , 22 ). B l i s a l s o much l e s s methylated than B2 but a p p a r e n t l y c o n t a i n s most o f the e - N - t r i m e t h y l l y s i n e p r e s e n t i n h i s t o n e H3. The m e t h y l a t i o n s i t e s o f H3 are summarized i n F i g . 14. (b) H i s t o n e H2B As noted i n s e c t i o n I , t h e r e was some apparent [ l l 4C] m e t h y l - l y s i n e i n c o r p o r a t i o n i n t o h i s t o n e H2B. There are l y s i n e s which c o u l d be methylated i n t h i s p r o t e i n a t p o s i t i o n s 5, 9, 10, 13, 14 e t c . , but automated sequencing o f t h i s h i s t o n e ( F i g . 10) gave a p r o f i l e o f r a d i o a c t i v i t y which Was l i k e t h a t f o r H3 (at p o s i t i o n s 4, 9, 27), which must have been p r e s e n t i n the H2B sample as a contaminant f o l l o w i n g p o o l i n g o f samples from the B i o - G e l P-10 column i n F i g . 3. (c) H i s t o n e H6 Histo n e H6. T h i s t r o u t - s p e c i f i c h i s t o n e showed a s m a l l amount o f i n c o r p o r a t i o n as [ 1 "*C]-methyl amino a c i d , and -46-3r No. of Degradations FIG. 10. Automated sequence a n a l y s i s o f [ l l fC]methyl l a b e l l e d t e s t i s h i s t o n e H2B. 7 mg of t e s t i s H2B (40,000 cpm) was se-q u e n t i a l l y degraded i n a Beckman 890 p r o t e i n sequencer, u s i n q v o l a t i l e reagents (122) . -47-3 4 5 c o n t a i n s a sequence around l y s i n e 4, arg - l y s - s e r (31 ), which i s homologous t o the sequence methylated i n h i s t o n e H3 a t p o s i t i o n s 9 and 27. For these reasons, we expected t h a t automated sequencing of the N - t e r m i n a l r e g i o n o f [ 1 "*C] methyl l a b e l l e d h i s t o n e H6 would show r a d i o a c t i v i t y r e l e a s e d on the f o u r t h d e g r a d a t i o n , a t l y s i n e 4. However, s e v e r a l attempts up to r e s i d u e 25 y i e l d e d no sharp peak of r e l e a s e d r a d i o -a c t i v i t y . Furthermore, as noted i n s e c t i o n I (b), t h e r e were no d e t e c t a b l e m e t h y l - l y s i n e s upon amino a c i d a n a l y s i s . Cd) H i s t o n e H4 The NH2-terminus o f t h i s p r o t e i n i s b l o c k e d by an a c e t y l group (12 ) and thus i s u n a v a i l a b l e f o r automated sequencing procedures; t h e r e f o r e , t r y p t i c p e p t i d e s o f m a l e y l a t e d [^C] methyl h i s t o n e H4 were an a l y z e d i n s t e a d . When the p e p t i d e s from such a d i g e s t were chromatographed on a Sephadex G-25 column, a s i n g l e r a d i o a c t i v e peak was o b t a i n e d ( F i g . 11), which a f t e r paper chromatography ( F i g . 12) proved t o be a s i n g l e p e p t i d e o f c o m p o s i t i o n l y s i n e + m e t h y l - l y s i n e (1.16), v a l i n e (1.10), l e u c i n e (1.00), a r g i n i n e (1.02). In F i g . 13 the amino a c i d sequence o f the p e p t i d e and r a d i o a c t i v i t y r e -l e a s e d a f t e r 3 Edman de g r a d a t i o n s are p r e s e n t e d . T h i s pep-t i d e corresponds to r e s i d u e s 20-23 of the p r o t e i n , w i t h m e t h y l a t i o n o c c u r r i n g a t l y s i n e 20, as was determined f o r c a l f thymus h i s t o n e H4 (12 ). L y s i n e 20 i s approximately -48-Fraction No., 10m/ FIG. 11. Sephadex G-25 chromatography of t r y p t i c p e p t i d e s o f m a l e y l a t e d , ['"cimethyl l a b e l l e d h i s t o n e H4. 70 mg of [ I 4 C ] -methyl l a b e l l e d h i s t o n e H4 c o n t a i n i n g 150,000 cpm, was m a l e y l -ated, d i g e s t e d w i t h t r y p s i n and chromatographed on a Sephadex G-2 5 column i n 25% a c e t i c a c i d . P r o t e i n was monitored by the n i n h y d r i n r e a c t i o n a t 570 nm. -49-s o l v e n t f r o n t C5 C4 C3 C2 Cl o r i g i n FIG. 12. Paper chromatography of t r y p t i c peptides of m a l e y l -ated H4. F o l l o w i n g Sephadex G-25 f r a c t i o n a t i o n of H4 t r y p t i c peptides ( F i g . 11), the [^C]methyl l a b e l l e d peak was pooled, concentrated and chromatographed on 3MM paper. The r a d i o a c t i v e peptide C5, was l o c a t e d by autoradiography. The other non-r a d i o a c t i v e peptides C1-C4, not v i s i b l e i n the autoradiogram, were l o c a t e d by n i n h y d r i n s t a i n i n g . -50-I 4 s o o o b 3 I i CJ O Me Lys Val Leu Arg 1 2 3 4 RESIDUE NUMBER FIG. 13. Sequence a n a l y s i s o f [1UC]methyl l a b e l l e d h i s t o n e H4. Manual Edman de g r a d a t i o n s were performed on the s i n g l e [ C ] -methyl l a b e l l e d p e p t i d e d e r i v e d from t r y p t i c d i g e s t i o n o f h i s -tone H4. A l i q u o t s o f the r e l e a s e d PTH d e r i v a t i v e s were counted i n Bray's s o l u t i o n . R a d i o a c t i v i t y r e l e a s e d i s p l o t t e d under the amino a c i d , i d e n t i f i e d by the s u b t r a c t i v e Edman procedure. -51-70% methylated, the r a t i o s of e-N-mono- to dimethyl-lysine being 1.6:1. There was no detectable t r i m e t h y l - l y s i n e . The sequence containing the methylation s i t e i n t e s t i s H4 i s shown i n F i g . 14. II I . (a) Histone Methylation i n the Different C e l l Types from  Developing Trout T e s t i s Having established that s p e c i f i c l y s y l residues of h i s -tones H3 and H4 were methylated - as had been described for other organisms ( 12, 17, 21,100) - we were next i n t e r -ested i n the re l a t i o n s h i p of t h i s histone methylation to the events of spermatogenesis. Louie and Dixon were able to separate, on bovine serum albumin gradients, d i f f e r e n t t e s t i s c e l l types and delineate t h e i r sequence of development as: spermatogonia 1°, 2° spermatocytes ->• early, l a t e spermatids mature sperm. Bio-chemically, DNA and histone synthesis stopped at the sperma-t i d stage, followed by the replacement of the histones by protamines i n t h i s c e l l type (93 ) . Was there a c o r r e l a t i o n between histone methylation and these c e l l types & biochemical events? To examine t h i s ques-t i o n , a t e s t i s was obtained 45 days a f t e r the s t a r t of i n j e c -tions with p i t u i t a r y extract. A c e l l suspension was made, incubated with [methyl- 3H]methionine and [ 1 U C ] l y s i n e , and the c e l l types were separated on a 1-3% bovine serum albumin -52-H3 Me Me Ac Ala-ArgThr Lys Glx Thr-Ala-Arg-Lys (Ser)-4 9 Ac Ac Thr Gly Gly Lys Ala Pro Arg -Lys-Glx-Leu-14 18 Ac Me AlaThr-LysAlaAla-Arg-Lys -Ser-Ala-Pro 23 27 Ac Ac Ac Ac-Ser-Gly Arg-Gly Lys Gly-Gly-Lys-Gly-Leu-Gly-Lys 5 8 12 Ac Me Gly-Gly Ala-Lys Arg-His-Arg- Lys-Val-Leu-Arg — 16 72o FIG. 14. The s i t e s o f a c e t y l a t i o n as determined by Candido and Dixon (22,36,40), and m e t h y l a t i o n of t r o u t t e s t i s h i s t o n e s H3 and H4. -53-g r a d i e n t ( 93,117). F r a c t i o n s of 7 ml were c o l l e c t e d . For each f r a c t i o n , c e l l s were counted on a hemocytometer, and TCA-tungstate p r e c i p i t a b l e r a d i o a c t i v i t y was determined. F i g u r e 15 shows t h a t the l a r g e r spermatocytes (3.5 mm/hr = Sv) and stem c e l l s (2.8 Sv) i n c o r p o r a t e f a r more [methyl- 3H]methionine on a per c e l l b a s i s , than do spermatids (1.5 S v ) . However, o n l y a p o r t i o n of t h i s TCA-tungstate p r e c i p i t a b l e i n c o r p o r a t i o n r e p r e s e n t s m e t h y l a t i o n o f h i s t o n e l y s y l r e s i d u e s ; some c o u l d d e r i v e from m e t h y l a t i o n of o t h e r macromolecules, or i n c o r p o r a t i o n of methionine i n t o c e l l p r o t e i n s . T h e r e f o r e , i n c o r p o r a t i o n s p e c i f i c a l l y i n t o h i s -tones was determined f o r the d i f f e r e n t c e l l types (pooled from the g r a d i e n t f r a c t i o n s i n F i g . 15). The r e s u l t s of a c i d e x t r a c t i o n o f the c e l l s d i r e c t l y i n t o s t a r c h g e l s l o t s , e l e c t r o p h o r e s i s and r a d i o a c t i v i t y a n a l y s i s of the h i s t o n e s are shown i n F i g . 16. I t i s apparent t h a t the l a r g e r d i p l o i d spermatocytes (pool a) and stem c e l l s (pool b ) , which are s t i l l a c t i v e i n DNA and h i s t o n e s y n t h e s i s (cf [ 1 hC]lysine i n c o r p o r a t i o n ) , account f o r most of the h i s t o n e m e t h y l a t i o n . The spermatid f r a c t i o n (pool c) shows much lower h i s t o n e m e t h y l a t i o n (20-30 f o l d lower, on a per c e l l b a s i s ) . T h i s accounts f o r the low h i s t o n e methyl i n c o r p o r a t i o n i n the u n f r a c t i o n a t e d c e l l p r o -f i l e , i n which spermatids predominate. As noted i n s e c t i o n I, almost a l l o f the [ 3H]methyl i n c o r p o r a t i o n occurs as e-N-methyl l y s i n e r a t h e r than as i n t e r n a l methionine; hence, the T— — — i 1 r FRACTION NO. (7.0ml) FIG. 15. Incorporation of L-[methyl- 3H]methionine and L - f ^ C ] -lysine into d i f f e r e n t c e l l types from trout t e s t i s . A t e s t i s c e l l suspension, from a f i s h 45 days a f t e r the s t a r t of matura-t i o n , was incubated with [ 3H]methionine and [lkC]lysine at 16°C for 2 hours and was fractionated at unit gravity on a 1-3% bovine serum albumin gradient (93,117). C e l l s were counted on a hemocytometer, and r a d i o a c t i v i t y was determined on an aliq u o t of each f r a c t i o n on a m i l l i p o r e f i l t e r , as described i n "Materials and Methods". Numbers over the arrows re f e r to sedimentation v e l o c i t y (Sv) i n millimeters per hour. -55-9 I 4 i r II Cells I 5 0 ° ( i b 9' W1A H1.H2A.H3 H» • Fl ii. : 1 '• J M c 1 2 i •5 a o « o 60 SO 100 DISTANCE FROM ORIGIN, CM FIG. 16. Synthesis and methylation of histones i n the d i f f e r e n t c e l l types from trout t e s t i s . C e l l s from regions a, b and c in Figure 15 were pooled, and [lhC]lysine and [ 3H]methyl incor-poration into histones was determined by starch gel e l e c t r o -phoresis as described i n "Materials and Methods". The histone bands are i d e n t i f i e d i n Figure 16a; P-H2A refer s to phosphoryl-ated H2A. -56-methyl i n c o r p o r a t i o n on the s t a r c h g e l i s s o l e l y i n t o h i s -11+ tones H3 and H4, whereas [ C ] l y s i n e i s i n c o r p o r a t e d i n t o a l l the h i s t o n e s . (b) Turnover o f methyl groups on h i s t o n e l y s y l r e s i d u e s We were next i n t e r e s t e d i n the t u r n o v e r o f h i s t o n e methyl groups i n t r o u t t e s t i s c e l l s . There are r e p o r t s t h a t h i s t o n e m e t h y l a t i o n i s a- s t a b l e m o d i f i c a t i o n (108), w h i l e o t h e r ob-s e r v a t i o n s show i t to be a dynamic p r o c e s s , w i t h methyl groups t u r n i n g over d u r i n g the c e l l c y c l e (100,107). The I k r e s u l t s o f experiments on [ C]methyl t u r n o v e r w i t h a mixed p o p u l a t i o n o f t r o u t t e s t i s c e l l s ( s i m i l a r t o t h a t used i n F i g u r e 15) i s shown i n F i g u r e 17. There i s no d e t e c t a b l e methyl t u r n o v e r up to 9 hours f o l l o w i n g the removal o f r a d i o a c t i v e l a b e l . P a r a l l e l i n c u b a t i o n s demonstrated c e l l v i a b i l i t y as measured by the a b i l i t y o f c e l l s t o i n c o r p o r a t e L-[ H ] a r g i n i n e i n t o 5% t r i c h l o r o a c e t i c a c i d - 0.2% t u n g s t a t e i n s o l u b l e m a t e r i a l ( F i g . 18). The l a c k o f d e t e c t a b l e t u r n -over of methyl groups makes i t u n l i k e l y t h a t d i f f e r e n t i a l methyl t u r n o v e r c o u l d account f o r the d i f f e r e n c e s i n h i s t o n e methyl i n c o r p o r a t i o n between the l a r g e r stem c e l l s and spermato-c y t e s v e r s u s spermatids. IV. K i n e t i c s of Histone H4 M e t h y l a t i o n The r e s u l t s presented i n s e c t i o n I I I i n d i c a t e t h a t h i s -tone m e t h y l a t i o n i s a r e l a t i v e l y s t a b l e m o d i f i c a t i o n , which occurs mainly i n the l a r g e d i p l o i d stem c e l l s and spermato-c y t e s , c e l l s which s t i l l a re s y n t h e s i z i n g DNA and h i s t o n e s . -57-4 6 8 Hours after Chase FIG. 17. Turnover of [^C] methyl groups i n trout t e s t i s h i s -tones. A t e s t i s c e l l suspension (5 x 10 8 c e l l s ) was incubated at 16°C for 2 hours with 100 yCi/ml of DL-[ 3H]lysine and 50 yCi/ml of L-[methyl- 1 "*C]methionine. The c e l l s were then c o l -lected by ce n t r i f u g a t i o n , resuspended i n 8 ml of isotope-free medium, and aliquots containing 5 x 10 1 • c e l l s were removed at in t e r v a l s and c o l l e c t e d on M i l l i p o r e glass f i b e r f i l t e r s . R adioactivity i n the histones was determined by starch gel electrophoresis; [ l l fC]methyl and [3H] ly s i n e incorporation were integrated over the histone region and the r a t i o of [ 1 !*C]-methyl to [ 3H]lysine incorporation was then calculated for each time sample. -58-K E C L u C O o I z FIG. 18. I n c o r p o r a t i o n o f [ 3 H ] a r g i n i n e i n t o t e s t i s n u c l e i w i t h t i m e - c e l l v i a b i l i t y . A f t e r i n c u b a t i o n of t e s t i s c e l l s f o r 2 hours With [ 3 H ] l y s i n e and [ m e t h y l - 1 4 C ] m e t h i o n i n e (see F i g . 17), an a l i q u o t of c e l l s was f u r t h e r i n c u b a t e d w i t h 50 yCi/ml of L - [ 3 H ] a r g i n i n e . At v a r y i n g .times, t o t a l r a d i o a c t i v i t y i n c e l l n u c l e i was determined by p r e c i p i t a t i o n w i t h TCA-tungstate on g l a s s f i b e r f i l t e r s . -59-We next examined the r e l a t i o n s h i p of t h i s methylation to the synthesis, a c e t y l a t i o n and phosphorylation of histone H4. H4 can be separated on urea-lactate starch gels into 10 d i f f e r e n t species (61), c o n s i s t i n g of unphosphorylated H4 with 0 to 4 e-N-acetyl-lysines (Ao to A 4) and slower mono-phosphorylated H4 with 0 to 4 acetate groups (PiAo to PiAit), as i n Figure 19. When Louie and Dixon l a b e l l e d histone H4 with [ 3H]lysine for increasing periods of time (84) , they found that newly synthesized, [ 3 H ] l y s i n e - l a b e l l e d H4 was f i r s t detectable only as the unphosphorylated, diacetylated species (A2). Only much l a t e r (after 16 hours) did [ 3H]lysine l a b e l appear i n the unmodified (Ao, Ai) and monophosphorylated (PiA 0, P1A1) species (Fig. 20). In other words, they could r e l a t e the pos i t i o n ( i . e . degree of acetylation) of histone H4 radio-a c t i v i t y on a starch g e l to the time a f t e r synthesis of the histone H4. On the basis of these r e s u l t s , they postulated for newly synthesized histone H4 an obligatory a c e t y l a t i o n and deacetylation c y c l e , which would be necessary for correct binding to DNA (84). As there i s evidence that histone methylation occurs l a t e i n the c e l l cycle a f t e r histone and DNA synthesis (100, 132), i t was of i n t e r e s t to see whether [ l l*C]methyl l a b e l l e d histone H4 appeared at the A2 p o s i t i o n c h a r a c t e r i s t i c of newly synthesized histone, or at the unmodified (A 0, A x) and monophosphorylated (PiA 0, P1A1) positions of "old" histone H4. For t h i s purpose, c e l l suspensions were l a b e l l e d with -60-FIG. 19. The acetylated and phosphorylated species of histone H4, separated by starch gel electrophoresis. Histone H4, pre-pared from trout t e s t i s by acid extraction, and carboxymethyl-c e l l u l o s e and Bio-Gel P-10 chromatography, was separated on a urea-lactate starch gel into the modified species. The bands are l a b e l l e d according to Sung and Dixon (61); subscript numbers refe r to number of e-N-acetyl (A) or phosphoryl (P) groups per histone H4 molecule. Bands were v i s u a l i z e d by staining the gel with Amido Black. -61-105 12 ia5 15 DISTANCE FROM ORIGIN, CM FIG. 20. [ 3 H ] l y s i n e i n c o r p o r a t i o n i n t o h i s t o n e H4 w i t h time, as described by Louie and Dixon (84) . T e s t i s c e l l s were i n c u -bated w i t h [ 3 H ] l y s i n e and a t v a r y i n g time i n t e r v a l s H4 was prepared. For longer i n c u b a t i o n s (>12 hours), [ 3 H ] l y s i n e was i n j e c t e d i n t e r p e r i t o n e a l l y i n t o l i v e t r o u t a t time = 0. The modified species of H4 were r e s o l v e d , and [ 3 H ] l y s i n e i n c o r p o r a -t i o n was determined w i t h u r e a - l a c t a t e s t a r c h g e l s . -62-(methyl- 1 1*C)methionine f o r up t o 12 hours, then h i s t o n e H4 was p u r i f i e d and e l e c t r o p h o r e s e d on u r e a - l a c t a t e s t a r c h g e l s . I t i s c l e a r from F i g u r e 21, t h a t u n l i k e newly s y n t h e s i z e d h i s t o n e H4, [ 1 I +C]methyl l a b e l l e d H4 appears immediately (t = 30 min) a t the unmodified (Ao, Ai) and monophosphorylated (PiAo, P i A i ) r e g i o n s , p o s i t i o n s c h a r a c t e r i s t i c o f h i s t o n e H4 a t l e a s t 16 hours a f t e r H4 s y n t h e s i s . A s m a l l amount ( l e s s than 10%) of [ ^ C j m e t h y l l a b e l i s i n c o r p o r a t e d not as e-N-m e t h y l l y s i n e but as [ 1 ''C] methionine i n t o newly s y n t h e s i z e d h i s t o n e H4 and c o u l d account f o r the low l e v e l s of r a d i o -a c t i v i t y i n the A 2 (new) s p e c i e s . With c o n t i n u e d i n c u b a t i o n (Figure 21), the p r o p o r t i o n o f [ x l |C]methyl l a b e l i n the mono-phospho r y l a t e d s p e c i e s i n c r e a s e s , a t r a n s i t i o n which p a r a l -l e l s the r e s u l t s o b t a i n e d by L o u i e and Dixon (84) f o r h i s -tone H4, from 16 hr to 1-3 days a f t e r s y n t h e s i s . -63-FIG. 21. [ 1 4C]methyl incorporation into histone H4 as a func-t i o n of time. Trout t e s t i s c e l l s were incubated with L-[methyl 1"C]methionine, at varying time i n t e r v a l s aliquots were re-moved, and histone H4 was prepared. The modified species of histone H4 were resolved and [ 1^C]methyl incorporation was determined, for each time point, with urea-lactate starch gels. -64-DISCUSSION There appear to be no g r e a t d i f f e r e n c e s i n the s i t e s of h i s t o n e m e t h y l a t i o n between t e r m i n a l l y d i f f e r e n t i a t i n g t r o u t t e s t i s c e l l s and o t h e r t i s s u e s s t u d i e d . The s i n g l e s i t e ( l y s i n e 20) of h i s t o n e H4 and the two major s i t e s ( l y s i n e s 9 and 27) o f h i s t o n e H3 are i d e n t i c a l t o those r e p o r t e d f o r c a l f thymus and oth e r t i s s u e s (15-20), except t h a t l a b e l l i n g w i t h [ 1 4 C ] m e t h y l groups allowed the d e t e c t i o n of a d d i t i o n a l minor s i t e s a t . l y s i n e 4 and p o s s i b l y l y s i n e 36 i n h i s t o n e H3. These minor s i t e s were a l s o observed (21) i n a comparative study o f N H 2 - t e r m i n a l sequences of h i s t o n e H3 from a cycad ( l y s i n e 4) and from c h i c k e n , shark, sea u r c h i n and m o l l u s c ( l y s i n e 36). The low l e v e l s o f [ ^ C l m e t h y l i n c o r p o r a t i o n i n t o h i s t o n e H6, without d e t e c t a b l e amounts o f e-N-methyl-lysines i n the p r o t e i n , c o u l d r e f l e c t some conta m i n a t i o n o f the p u r i f i e d h i s -tone H6 by t r a c e amounts o f l a b e l l e d h i s t o n e H3 or H4. Auto-mated p r o t e i n sequencing d i d not however g i v e a p r o f i l e o f r a d i o a c t i v i t y r e l e a s e d which i s c h a r a c t e r i s t i c o f h i s t o n e H3. 1 4 A l t e r n a t i v e l y , [ C]methyl i n c o r p o r a t i o n c o u l d r e f l e c t t r a c e amounts o f m e t h y l a t i o n s i m i l a r t o t h a t observed f o r 3-methyl-h i s t i d i n e i n a v i a n e r y t h r o c y t e h i s t o n e H5 by Gershey e t a l . (43). DeLange, Smith and co-workers have noted (9,10) the sim-i l a r i t i e s among methylated sequences: -x-Arg-Lys-x- (common to h i s t o n e H4, H3) and -A l a - A r g - L y s - S e r - ( f o r both major s i t e s o f h i s t o n e H3). However, h i s t o n e H6 i s not s i g n i f i c a n t l y methylated i n the sequence -Arg-Lys-Ser around l y s i n e 4. T h i s -65-suggests that e i t h e r (i) recognition by the methylase re-quires s t r u c t u r a l features a d d i t i o n a l to those i n the t r i -peptide-Arg-Lys-Ser; or ( i i ) the location or structure of histone H6 makes i t inaccessible to methylation. P u r i f i c a t i o n and studies on the substrate s p e c i f i c i t y of the histone methyl-ase a c t i v i t y present i n trout t e s t i s n u c l e i might resolve t h i s question. There i s as yet no information on the function of histone H6. This minor (1% of t o t a l ) , t r o u t - s p e c i f i c histone resembles histone H2B i n s i z e , and l y s i n e to arginine r a t i o (19), but i s l i k e Hi i n i t s e x t r a c t a b i l i t y from chromatin, i t s high content of l y s i n e , alanine and p r o l i n e , and i t s postsynthetic modifications (H6 can be phosphorylated, but i s neither a c e t y l -ated nor methylated)"*". None of the other histones contained s i g n i f i c a n t amounts of methyl-lysines except histone H2B, which was l i k e l y contam-inated with H3. Histones H3 and H4 are highly conserved i n amino acid sequence and methylation s i t e s i n d i f f e r e n t species, yet there are differences i n the extent of methylation of lysines at those s i t e s (the most dramatic being the complete absence of methyl-lysi n e i n pea histone H4). The resultant heterogeneity of histone molecules with respect to number of methyl groups, as pointed out by A l l f r e y (3), makes i t d i f f i c u l t to assign a generalized or universal function f o r histone methylation. ^ B.M. Honda, unpublished r e s u l t s -66-H i s t o n e M e t h y l a t i o n i n D i f f e r e n t C e l l Types. When t e s t i s c e l l s are i n c u b a t e d w i t h [methyl- 3H]methionine and s e p a r a t e d a t one g r a v i t y , methyl i n c o r p o r a t i o n i n t o h i s t o n e s occurs predom-i n a n t l y i n the l a r g e spermatocytes and stem c e l l s ( F i g . 15 and 16). T h i s m e t h y l a t i o n i s absent from spermatids and so probably has no r o l e i n the displacement o f h i s t o n e s by p r o t -amine, a p r o c e s s which may r e q u i r e h i s t o n e a c e t y l a t i o n (78). U n l i k e the spermatids, the d i p l o i d spermatocytes and stem c e l l s are s t i l l a c t i v e l y s y n t h e s i z i n g DNA and h i s t o n e s and undergoing c e l l d i v i s i o n (93). I t i s p o s s i b l e then t h a t h i s t o n e m e t h y l a t i o n has some r o l e i n these p r o c e s s e s , o r i n the condensation of chromatin p r i o r to m i t o s i s (100,108). Turnover o f H i s t o n e Methyl Groups. The l a c k of d e t e c t a b l e t u r n o v e r o f h i s t o n e methyl groups ( F i g . 18) makes i t u n l i k e l y t h a t d i f f e r e n t i a l methyl t u r n o v e r c o u l d account f o r e i t h e r (i) the h e t e r o g e n e i t y o f h i s t o n e molecules w i t h r e s p e c t to number of methyl groups or ( i i ) the d i f f e r e n c e s i n h i s t o n e methyl i n c o r p o r a t i o n between the l a r g e r stem c e l l s and sperm-a t o c y t e s v e r s u s spermatids. I t i s p o s s i b l e t h a t a v e r y slow t u r n o v e r of methyl groups occurs over a c e l l c y c l e , which f o r t r o u t t e s t i s i s 6-7 days (93), much l o n g e r than the time o f the experiment. However, complete t u r n o v e r o f methyl groups d u r i n g a c e l l c y c l e appears u n l i k e l y . A methyl t u r n o v e r r a t e o f 2% per hour i n Hela c e l l s was e s t i m a t e d by Borun e t a l . (107), but t h i s e s t i m a t e was based -67-14 3 • on r a t i o s of C to H i n c o r p o r a t i o n which were widely s c a t t e r e d about the mean r a t e . Moreover, the p u t a t i v e p r o t e i n "demethyl-ase" of Paik and Kim (109), which might be r e s p o n s i b l e f o r methyl group turnover, has not been shown t o a c t on chromatin-a s s o c i a t e d h i s t o n e s , and i t s s u b s t r a t e s and products have not yet been c l e a r l y i d e n t i f i e d . In t r o u t t e s t i s c e l l s then, h i s t o n e m e t h y l a t i o n i s a r e l a t i v e l y s t a b l e m o d i f i c a t i o n . K i n e t i c s of Histone H4 M e t h y l a t i o n . Louie and Dixon (84) demonstrated t h a t newly syn t h e s i z e d H4 underwent an o b l i g a -t o r y r a p i d a c e t y l a t i o n to the d i a c e t y l a t e d form A2, f o l l o w e d by a slower a c e t y l a t i o n , p h o s p h o r y l a t i o n , and d e a c e t y l a t i o n pathway. The r e s u l t s of [^C] methyl l a b e l l i n g of h i s t o n e H4 w i t h time a l l o w us t o place H4 met h y l a t i o n i n t o t h i s scheme of H4 metabolism. Since the behaviour of [ 1 I #C]methyl l a b e l l e d h i s -tone H4 ( F i g . 21) p a r a l l e l s t h a t of h i s t o n e H4 some 16 hours t o 1-3 days a f t e r s y n t h e s i s , t h i s suggests t o us t h a t p o s t -s y n t h e t i c events i n h i s t o n e H4 metabolism c o n s i s t of a c e t y l a -t i o n and d e a c e t y l a t i o n (up to 12-16 hours), f o l l o w e d by methyl-a t i o n and pho s p h o r y l a t i o n . I t i s u n l i k e l y t h a t m e t h y l a t i o n occurs on a sm a l l pool of h i s t o n e H4 behaving d i f f e r e n t l y from the m a j o r i t y . Such a pool (which, f o r example might bypass a c e t y l a t i o n ) would be at most a few percent of the t o t a l , judging from the r e s u l t s of Louie and Dixon (84), whereas me t h y l a t i o n occurs on a t -68-l e a s t 70% of h i s t o n e H4 molecules i n t h i s t i s s u e ( s e c t i o n I I ) . Furthermore, the s h i f t o f methyl l a b e l t o p h o s p h o r y l a t e d s p e c i e s shows a good temporal correspondence w i t h the behaviour of h i s t o n e H4 16 hours to 1-3 days a f t e r s y n t h e s i s . These r e s u l t s c o r r e l a t e w e l l w i t h the work of T i d w e l l e t a l . (132) and Paik and Kim (100), showing t h a t h i s t o n e m e t h y l a t i o n occurs l a t e i n the c e l l c y c l e , a f t e r DNA and h i s t o n e s y n t h e s i s and p r i o r t o chromatin condensation and m i t o s i s . I f the same a c e t y l a t i o n , d e a c e t y l a t i o n , m e t h y l a t i o n time course i s a p p l i c a b l e to h i s t o n e H3, then the d i f f e r e n c e s i n j. e x t e n t of m e t h y l a t i o n and a c e t y l a t i o n between H3 p e p t i d e s B l and B2 (with i d e n t i c a l c o m p o s i t i o n see T a b l e I I I ) might be e x p l a i n e d . P e p t i d e B l (Table I I I ) would be d e r i v e d from r e c e n t l y s y n t h e s i z e d , a c e t y l a t e d and undermethylated h i s t o n e H3 molecules, whereas p e p t i d e B2 would d e r i v e from o l d e r , more methylated m o l e c u l e s . The presence o f r e l a t i v e l y h i g h ' l e v e l s o f e - N - t r i m e t h y l - l y s i n e i n B l i s d i f f i c u l t t o i n t e r p r e t , however. What i s the f u n c t i o n o f h i s t o n e m e t h y l a t i o n ? No c l e a r answer emerges. From the o b s e r v a t i o n s — (i) t h a t the methyla-t i o n s i t e s ( l y s i n e s 9,27) o f h i s t o n e H3 are a d j a c e n t t o the p h o s p h o r y l a t i o n sites"*" ( a l s o i n f e r r e d from r e f . 38) a t s e r i n e s 10 and 28; ( i i ) t h a t h i s t o n e m e t h y l a t i o n i s a s s o c i a t e d w i t h the l a r g e r d i v i d i n g c e l l types and ( i i i ) t h a t h i s t o n e H4 phos-p h o r y l a t i o n o c c u r s c o i n c i d e n t l y w i t h , or a f t e r m e t h y l a t i o n — B.M. Honda, unpublished r e s u l t s -69-i t i s p o s s i b l e t h a t h i s t o n e m e t h y l a t i o n i s a n e c e s s a r y p r e l u d e to p h o s p h o r y l a t i o n and subsequent chromatin c o n d e n s a t i o n and m i t o s i s . T h i s m e t h y l a t i o n may i n v o l v e changes i n charge, conformation or h y d r o p h o b i c i t y of the h i s t o n e , a l l o w i n g i n t e r -a c t i o n s w i t h a h i s t o n e phosphokinase or o t h e r m o l e c u l e s . S i n c e H3 and H4 i n t e r a c t w i t h each o t h e r (32-34) i n tetramers t o g i v e a s u b u n i t - l i k e s t r u c t u r e to chromatin - (see P a r t B, t h i s t h e s i s ) , m e t h y l a t i o n of these 2 h i s t o n e s might a l s o induce s t r u c -t u r a l changes i n chromatin r e q u i r e d f o r c e l l d i v i s i o n . F u r t h e r experiments on the i s o l a t i o n , c h a r a c t e r i z a t i o n and r e g u l a t i o n of the enzymes r e s p o n s i b l e f o r h i s t o n e mod-i f i c a t i o n s should c l a r i f y the r o l e o f h i s t o n e m e t h y l a t i o n i n these c e l l u l a r e v ents. PART B: CHROMATIN SUBUNIT STRUCTURE - 7 0 -INTRODUCTION The s t r u c t u r e and f u n c t i o n o f c h r o m a t i n — t h a t complex o f DNA, h i s t o n e s , n o n - h i s t o n e p r o t e i n s and s m a l l amounts o f RNA i n t h e c e l l n u c l e u s — has l o n g been a m y s t e r y . As n o t e d i n P a r t A o f t h i s t h e s i s , the h i s t o n e s a r e s m a l l b a s i c p r o t e i n s p r e s e n t i n e q u a l w e i g h t w i t h t h e DNA. T h e r e a r e 5 major c l a s s e s o f t h e s e p r o t e i n s , t h o u g h t t o be i m p o r t a n t i n m a i n -t a i n i n g c h r o m a t i n s t r u c t u r e and p o s s i b l y " c o a r s e " c o n t r o l o f gene a c t i v i t y . U n l i k e t h e h i s t o n e s , t h e n o n - h i s t o n e p r o t e i n s (NHP) o f c h r o m a t i n a r e a h e t e r o g e n e o u s p o p u l a t i o n w i t h many d i f f e r e n t s p e c i e s up t o 100,000 d a l t o n s i n m o l e c u l a r w e i g h t . (For a r e v i e w , see (133-135.) R e p o r t s on NHP s t o i c h i o m e t r y v a r y f rom r e p o r t s o f e q u a l w e i g h t w i t h DNA (135) t o o n l y 3% by w e i g h t (136) . I n t h e l a t t e r c a s e t h e a u t h o r s do n o t d i s -p u t e the n e c e s s i t y f o r NHP t o r e g u l a t e gene a c t i v i t y ; r a t h e r , t h e y q u e s t i o n r e p o r t e d l a r g e amounts o f NHP as p o s s i b l e mem-b r a n e (137) o r c y t o p l a s m i c c o n t a m i n a n t s (138) . The f u n c t i o n s o f NHP a r e u n c l e a r . These p r o t e i n s e x h i b i t t i s s u e and s p e c i e s s p e c i f i c i t y (134) and have been d e s c r i b e d as p r e s e n t i n i n c r e a s e d amounts i n g e n e t i c a l l y a c t i v e t i s s u e s (134) . The i n c r e a s e d s y n t h e s i s , p h o s p h o r y l a t i o n o r t u r n o v e r o f s p e c i f i c NHP has i m p l i c a t e d them i n t h e i n d u c t i o n o f gene a c t i v i t y (134) , as f o r example i n r e s p o n s e t o s t e r o i d hormones (139-141) . I t has a l s o been r e p o r t e d t h a t t h e NHP, n o t h i s t o n e s , c o n f e r t i s s u e - s p e c i f i c t r a n s c r i p t i o n a c t i v i t y t o r e c o n s t i t u t e d c h r o m a t i n s (142) . The NHP may a l s o r e g u l a t e -71-c e l l d i v i s i o n ; the s y n t h e s i s and p h o s p h o r y l a t i o n of d i f f e r e n t NHP a t d i f f e r e n t stages o f the c e l l c y c l e have been d e s c r i b e d i n support o f t h i s h y p o t h e s i s (143). F i n a l l y , some NHP must be n u c l e a r enzymes (134) or p o s s i b l y s t r u c t u r a l components o f chromatin. In what k i n d o f s t r u c t u r e are DNA and p r o t e i n s assembled i n chromatin? U n t i l r e c e n t l y , the most w i d e l y accepted model of chromatin came from the X-ray d i f f r a c t i o n s t u d i e s of W i l k i n s (144) and o t h e r s (145). They observed r e g u l a r l y spaced d i f f r a c t i o n r i n g s from chromatin f i b e r s . From t h i s they i n t e r p r e t e d chromatin as a DNA double h e l i x coated w i t h p r o t e i n s and wound i n a l a r g e r c o i l o r s u p e r h e l i x (144). The arrangement — r e g u l a r , random or o t h e r — of p r o t e i n s on the DNA remained u n c e r t a i n . Then i n 1973, Hewish and Burgoyne (146) noted t h a t an endogenous n u c l e a s e i n r a t l i v e r n u c l e i c l e a v e d the DNA, not randomly t o a l l s i z e s , but r a t h e r t o d i s c r e t e fragments, m u l t i p l e s o f a u n i t l e n g t h . T h i s suggested t o them t h a t the p r o t e i n s were arranged i n an o r d e r e d , r e p e t i t i v e manner al o n g the DNA, p r o t e c t i n g the DNA from n u c l e a s e d i g e s t i o n . N o l l then found (147) t h a t treatment o f i n t a c t r a t l i v e r n u c l e i w i t h the endogenous n u c l e a s e o r commercially a v a i l a b l e m i c r o -c o c c a l n uclease c l e a v e d 85% o f the DNA t o fragments 200 base p a i r s l o n g o r m u l t i p l e s t h e r e o f . L o u i e (148) o b t a i n e d s i m i l a r r e s u l t s w i t h polyoma n u c l e o p r o t e i n . N o l l (147) c o u l d a l s o i s o l a t e , from n u c l e a s e - t r e a t e d n u c l e i , i n t a c t 11. 2S "chromatin s u b u n i t s " , c o n t a i n i n g h i s t o n e s and non-histone p r o t e i n s -72-attached to stretches of DNA 200 base pa i r s long. Van Holde and coworkers (149) had e a r l i e r described d i s c r e t e p a r t i c l e s r e s u l t i n g from micrococcal nuclease digestion of chromatin. From t h e o r e t i c a l considerations, for each histone Hi molecule there could be two each of H2A, H2B, H3 and H4, and an equal weight of DNA which corresponds to a length of roughly 200 base pairs as observed. Electron micrographs showing bead-l i k e regions spaced along chromatin (150) supported such a picture of subunits of nuclease-resistant, protein-bound DNA a l t e r n a t i n g with nuclease-sensitive, DNA spacer regions. Romberg then extended such r e s u l t s , along with his own data on crosslinked histone oligomers (151), into a theory of chromatin structure (152) based on a repeating u n i t of 200 DNA base p a i r s and two of each of the histones (except f o r histone I ) . A s i m i l a r model was proposed by Van Holde and coworkers (153) . N o l l also observed that when chromatin i n s i t u was d i -gested with DNase I, a regular s e r i e s of single-stranded DNA fragments, multiples of 10 bases long, were obtained (154). His work strongly suggested that DNA i s accessible to DNase, on the outside of the chromatin subunit, the structure of which contains some i n t e r n a l r e p e t i t i v e elements. D i f f e r e n t DNA fragments, smaller than 200 base pairs long, had been report-ed by Van Holde (149), Axel et a l . (155), and Weintraub & coworkers (156) when i s o l a t e d chromatin, as opposed to i n t a c t n u c l e i , were -73-t r e a t e d w i t h m i c r o c o c c a l n u c l e a s e . Such r e s u l t s a l s o p o i n t e d to some r e g u l a r repeat s t r u c t u r e f o r chromatin, based on " s p e c i f i c c o n t a c t s between p r o t e i n and n u c l e i c a c i d which a r i s e from s t r u c t u r a l p r o p e r t i e s o f the h i s t o n e s " (156). T h i s c o u l d have i n v o l v e d the c r o s s l i n k i n g of DNA by the ex-posed N - t e r m i n i of a t r y p s i n - r e s i s t a n t h i s t o n e complex (156), but i t was d i f f i c u l t t o r e l a t e these n u c l e a s e d i g e s t i o n p r o-ducts to those o b t a i n e d by N o l l (147). At the time t h i s i n v e s t i g a t i o n was s t a r t e d then, we were i n t e r e s t e d i n examining the subun i t s t r u c t u r e o f chromatin from t r o u t t e s t i s . T r o u t t e s t i s i s a good t i s s u e .for the study of such chromatin s u b u n i t s s i n c e : (i) l a r g e q u a n t i t i e s of chromatin r e l a t i v e l y f r e e of c y t o p l a s m i c contamination are e a s i l y prepared; ( i i ) the su b u n i t s r e p r e s e n t one approach to st u d y i n g changes i n chromatin s t r u c t u r e when the h i s t o n e s are r e p l a c e d by protamine d u r i n g sperm development; and ( i i i ) the s u b u n i t s p r o v i d e a model s u b s t r a t e f o r the study o f the enzymatic m o d i f i c a t i o n s o f h i s t o n e s i n t r o u t t e s t i s . In P a r t B of t h i s t h e s i s , i t i s shown t h a t nuclease treatment o f n u c l e i from t r o u t t e s t i s a t e a r l y (histone) stages o f development g i v e s s i m i l a r DNA fragments and 1.1 S chromatin s u b u n i t s t o those r e p o r t e d i n oth e r s t u d i e s (146-148). T h i s data f u r t h e r supports the model of e u k a r y o t i c chromatin as 200 base p a i r long segments of DNA covered w i t h p r o t e i n s . These s u b u n i t s would be separated by n u c l e a s e - s e n s i t i v e spacer DNA. - 7 4 -T e s t i s c o n s i s t i n g predominantly o f e a r l y spermatids (m e i o t i c t i s s u e , c o n t a i n i n g mainly n u c l e o h i s t o n e ) g i v e s s i m i l a r y i e l d s of DNA fragments and 11S s u b u n i t s . L a t e r stage t e s t i s (protamine has r e p l a c e d the h i s t o n e s ) however, g i v e s no DNA fragments or l i s s u b u n i t s . T h i s presumably r e f l e c t s l a r g e d i f f e r e n c e s i n s t r u c t u r e between n u c l e o p r o t -amine and n u c l e o h i s t o n e . F i n a l l y , the composition and some p r o p e r t i e s o f t e s t i s chromatin s u b u n i t s are r e p o r t e d , and d i s c u s s e d i n terms of more r e c e n t f i n d i n g s . -75-MATERIALS.AND METHODS I. Chemicals and A b b r e v i a t i o n s (a) Chemicals A l l chemicals obtained commercially were of the highest p u r i t y or reagent grade. S p e c i a l reagents were obtained as f o l l o w s : Bovine c a t a l a s e (E.C. 1.11.1.6) from Worthington; m i c r o c o c c a l nuclease (E.C. 3.1.4.7) from Sigma; acrylamide from Matheson, Coleman and B e l l ; TEMED (N,N,N*,N'-tetramethyl-ethylenediamine) from Canal I n d u s t r i a l Corp; N, N'-methy1ene— bi s a c r y l a m i d e and " S t a i n s a l l " from Eastman Kodak. (b) A b b r e v i a t i o n s EDTA: e t h y l e n e d i a m i n e - t e t r a a c e t i c a c i d SDS: sodium dodecyl s u l f a t e S t a i n s a l l : l - e t h y l - 2 - [ 3 - ( 1 - e t h y l n a p h t h [ 1 , 2 a ] - t h i a z o l i n -2-ylidene)-2-methylpropenyl]naphtho[1,2a]-thiazolium bromide TMKS b u f f e r : T r i s - H C l (50 mM, pH 7.4), MgCl 2 (1 mM), KC1 (25 mM), and sucrose (0.25 M). TMKSM b u f f e r : TMKS b u f f e r made 15 mM i n 2-mercaptoethanol : i . P r e p a r a t i o n and Nuclease D i g e s t i o n of N u c l e i or I s o l a t e d Chromatin N a t u r a l l y maturing t r o u t t e s t e s (obtained from Sun V a l l e y Trout Farm, M i s s i o n , B.C.) were v i g o r o u s l y homogenized i n TMKSM b u f f e r , and n u c l e i were p e l l e t e d a t 1,000 g f o r 10 min. A f t e r two more washings w i t h the same b u f f e r , 5 - 10 x 10 8 -76-nuclei/ral were incubated at 37°C for 1-10 min with 500 A 26o units/ml of micrococcal nuclease i n TMKSM buffer made 1 mM in CaCl2. In l a t e r preparations of n u c l e i , 2-mercaptoethanol was omitted from the buffers. Chromatin was prepared by washing nu c l e i further with 0.15 M NaCl, 20 mM EDTA, and then three times i n 10 mM T r i s pH 8.0. Digestion of chromatin (20 A 2 6 0 of DNA/ml) was performed at 37°C i n 10 mM T r i s , 0.1 mM C a C l 2 , with 300 units/ml of micrococcal nuclease. I I I . Determination of Developmental Stages (histone:protamine ratio) of Testes Nuclei prepared as above were extracted with 0.4 N H2SCH and basic proteins were p r e c i p i t a t e d with 3 volumes of ethanol at -20°C. Proteins were electrophoresed on urea-15% poly-acrylamide gels (157) stained with Coomassie Blue and scanned at 600 nm with a G i l f o r d spectrophotometer [see Materials and Methods, section VIII (b) for acrylamide gel e l e c t r o -phoresis] . IV. Preparation of "Chromatin Subunits" from Digested Nuclei A f t e r treatment with micrococcal nuclease, n u c l e i were c o l l e c t e d at 1000 g, resuspended vigorously i n 0.2 mM EDTA, and spun at 12,000 g f o r 30 min as f i r s t described by N o l l (147). The supernatant, containing 10-200 A 2 6 o / m l of chromatin subunits, was then layered on a 10-30% sucrose-0.2 mM EDTA - 7 7 -g r a d i e n t (12 ml) and spun a t 3 6,000 rpm f o r 12-14 h o u r s i n a Beckman SW40-Ti r o t o r . F r a c t i o n s o f 0.4 m l were c o l l e c t e d by p u n c t u r i n g the g r a d i e n t t u b e , f o l l o w e d by upward d i s -placement o f the g r a d i e n t w i t h 60% s u c r o s e . In e a r l i e r e x p e r i m e n t s , s u b u n i t s were spun a t 25,000 rpm f o r 18 h r i n a Beckman SW27 r o t o r , and twenty d r o p f r a c -t i o n s c o l l e c t e d from t h e tube bot tom by s i p h o n i n g . B o v i n e c a t a l a s e , and E . c o l i 3 - g a l a c t o s i d a s e , s e d i m e n t i n g i n a s e p -a r a t e t u b e , were found t o be c o n v e n i e n t markers f o r t h e 11S and 16S r e g i o n s r e s p e c t i v e l y . F o r a d i g e s t c o n t a i n i n g p r e d o m i n a n t l y monomers, n u c l e i f r o m 2 grams o f t e s t i s were d i g e s t e d i n 5 ml o f TMKS-CaCl2 b u f f e r , 8 m i n , w i t h 500 u n i t s / m l o f m i c r o c o c c a l n u c l e a s e . A f t e r d i g e s t i o n and c e n t r i f u g a t i o n , n u c l e i were r e s u s p e n d e d i n 2 m l o f 0.2 mM EDTA. T h i s gave a p p r o x i m a t e l y 80-100 A 2 6 0 / ml o f s u b u n i t s a f t e r the c e n t r i f u g a t i o n a t 12,000 g , 30 m i n . V . A n a l y s i s o f DNA Fragments Produced by N u c l e a s e D i g e s t i o n (a) I s o l a t i o n o f DNA fragments DNA was p r e p a r e d from m i c r o c o c c a l n u c l e a s e - t r e a t e d n u c l e i o r c h r o m a t i n , o r f rom i s o l a t e d c h r o m a t i n s u b u n i t s , by p h e n o l e x t r a c t i o n (147) . Samples i n 1% SDS, M N a C l , 20 mM EDTA were e x t r a c t e d t w i c e w i t h p h e n o l . DNA was p r e c i p i -t a t e d from the r e s u l t i n g aqueous phase w i t h 2 volumes o f e t h a n o l a t - 2 0 ° C . -78-(b) Quantitation of DNA In t h i s procedure described by Burton (158), DNA samples (10-300 yg/ml) were made 0.5 N i n p e r c h l o r i c acid and heated at 70°C for 15 min. After cooling, a 0.33 ml aliq u o t was reacted with 0.67 ml of diphenylamine reagent (1.5 g diphenyl-amine i n 100 ml acetic acid plus 1.5 ml H2SOO to which acetaldehyde had been fr e s h l y added (0.1 ml of 1.6% aqueous acetaldehyde per 20 ml of reagent). Colour was developed at 30°C for 16-20 hr, and the absorbance at 565 nm was monitored against a blank, and standard c a l f thymus DNA samples of known concentration. (c) Non-denaturing 2.5% polyacrylamide gel e l e c t r o - phoresis of DNA In t h i s technique described by Loening (159), the follow-ing volumes of stock so l u t i o n s : - 5 ml of lOx concentrated TEA buffer (TEA buffer = 0.04 M T r i s - a c e t i c acid pH 7.8, 2 mM EDTA, 0.02 M sodium acetate), 6.25 ml of 20% acrylamide solution (acrylamide: N,N'-methylenebisacrylamide, 19:1) -were combined with 38 ml of water and deaerated under vacuum. Then 40 mg of ammonium persulfate i n 0.7 ml of H 2 O , and 40 y l of TEMED (N,N,N',N'-tetramethylethylenediamine) were added, and the acrylamide was polymerized under isobutanol i n 7 cm tubes. , -79-Samples of 5-100 yg of DNA i n 10% sucrose, 0.005% bromo-phenol blue were electrophoresed i n TEA b u f f e r at 4°C f o r 1-lh hours at 5 m i l l i a m p e r e s / g e l . Gels were s t a i n e d o v e r n i g h t i n S t a i n s a l l (0.005% i n 50% formamide), then destained i n water by b r i e f exposure t o l i g h t . Gels were then scanned at 550 nm i n a G i l f o r d spectrophotometer. (d) Denaturing 99% formamide, 6% polyacrylamide g e l separation  of DNA For these g e l s d e s c r i b e d by Staynov e t a l . (160), 99% formamide i s s t i r r e d w i t h Dowex 50W-X8 (3 g/100 ml) f o r 1 hour, and i s then f i l t e r e d and used the same day. For a 6% acrylamide (85:15) g e l , 2.04 g of acrylamide, 0.36 g of N,N'-methylenebisacrylamide and 80 y l of TEMED were d i s s o l v e d i n 40 ml of formamide, which was then f i l t e r e d . Then 50 mg of ammonium p e r s u l f a t e , d i s s o l v e d i n 0.8 ml o f M sodium phosphate pH 7.0, was added to make the g e l 20 mM i n phosphate and 0.12% ammonium p e r s u l f a t e . A 15 x 15 x 0.15 cm s l a b g e l , sealed a t the bottom w i t h p l a s t i c spacer and v a s e l i n e , was then poured and polymerized. RNA and DNA samples were d i s s o l v e d i n formamide ( c o n t a i n -i n g 20 mM sodium phosphate pH 7.0, 20% sucrose, 0.005% bromo-phenol b l u e ) , heated to 70°C, and electrophoresed i n formamide-20 mM phosphate a t up t o 100 v o l t s , 10 m i l l i a m p e r e s f o r 12-16 hours. Gels were s t a i n e d i n S t a i n s a l l overnight and destained i n l i g h t , , as d e s c r i b e d above. -80-VI. Sepharose 2B Chromatography o f Chromatin Subunits Chromatin s u b u n i t s were prepared ( M a t e r i a l s and Methods, s e c t i o n I I I ) i n 0.2 mM EDTA, and chromatographed on a 2.5 x 40 cm Sepharose 2B (Pharmacia) column, e q u i l i b r a t e d and run a t 4°C i n 5 mM T r i s - H C l , 0.2 mM EDTA, pH 7.6. V I I . V e l o c i t y Sedimentation Experiments on Subunits When a c e n t r i f u g a l f i e l d i s a p p l i e d t o a un i f o r m s o l u -t i o n o f macromolecules i n the se c t o r - s h a p e d c e l l o f the Beckman Model E A n a l y t i c a l U l t r a c e n t r i f u g e , the r e g i o n near the meniscus (top) o f the c e l l i s p r o g r e s s i v e l y c l e a r e d o f molecules w i t h time. From the v e l o c i t y o f t h i s moving bound-ary o f molecules, one can determine the s e d i m e n t a t i o n co-dr 1 e f f i c i e n t o f the mo l e c u l e s , S = v r . — 2 — , where r i s the a t w r displacement from the r o t o r c e n t e r ; ^ i s then the v e l o c i t y , and w 2r i s the c e n t r i f u g a l f i e l d s t r e n g t h . Rearranging the 1 ? . . ; e q u a t i o n , t o d r • — = w s • d t , and i n t e g r a t i n g , g i v e s the r e l a t i o n s h i p In ^ = w 2s ( t - t o ) . From the s l o p e , w 2s, o f a p l o t o f In r / r o vs t - t o , one can c a l c u l a t e a v a l u e o f s, o b t a i n e d a t temperature T i n b u f f e r b. To c o r r e c t the s v a l u e t o a s t a n d a r d S20 / W (s v a l u e a t 20°C i n d i s t i l l e d w a t e r ) , the e q u a t i o n used i s : S 2 0 , w = {l-VPWo,w ^ . s where V i s the p a r t i a l s p e c i f i c volume, p the s o l u t i o n d e n s i t y and n the s o l u t i o n v i s c o s i t y . -81-Samples of subunits i n 20 mM T r i s , 0.2 mM EDTA, pH 7.0 were sedimented at 36,000 or 40,000 rpm i n a 1.61 cm sector c e l l at 4°C. The moving boundary was monitored to give a concentration vs distance p r o f i l e at 15-20 min i n t e r v a l s i n eithe r of two ways as follows; (i) Scanner o p t i c s . Subunits are sedimented at a con-centration of 0.8 A26o/ml. UV l i g h t (A = 260 nm) i s passed through the c e l l and darkens a photographic plate. Absorb-ance, which w i l l r i s e from 0 at the meniscus, to 0.8 at the boundary, i s measured by densitometric scanning of the photo-graphic p l a t e . This gives an absorbance vs distance p r o f i l e at each time point. ( i i ) Schlieren o p t i c s . Subunits are sedimented at 60 A2 6o/ml. At the moving boundary, there i s a change i n re-f r a c t i v e index (An) such that p a r a l l e l l i g h t rays passing through i t are deflected (as i n a prism). In the ultraeen-t r i f u g e , p a r a l l e l l i g h t i s passed through the c e l l , and then through a series of lenses and a diagonal s l i t ( i n c l i n e d at angle 0 ) . Light rays passing above or below the molecule boundary i n the c e l l are not deflected (An = o), and give a s t r a i g h t l i n e on a photographic pl a t e . However, l i g h t rays passing through the boundary are deflected (An > o), and are displaced sideways on the photographic pla t e , i n a displacement pro--82-p o r t i o n a l t o An. The r e s u l t on the photographic p l a t e at the p o s i t i o n of the boundary, i s a peak which corresponds mathematically t o the f i r s t d e r i v a t i v e ( g ^ ) o f t h e c Y_s r p r o f i l e o b t a i n a b l e w i t h scanner o p t i c s . In experiments w i t h s u b u n i t s , 0 = 50°. V I I I . P r o t e i n Composition of Chromatin Subunits (a) I s o l a t i o n of p r o t e i n s from subunits or whole chromatin Se v e r a l methods were used to remove p r o t e i n s from subunit DNA. (i ) A l i q u o t s of chromatin subunits were made 1 t o 5% i n SDS and heated to 100°C to r e l e a s e p r o t e i n s p r i o r to SDS g e l e l e c t r o p h o r e s i s . Subunits were sometimes t r e a t e d w i t h DNase I (5 yg/sample) p r i o r to SDS e x t r a c t i o n , t o a v o i d DNA c l o g g i n g of polyacrylamide g e l s . ( i i ) Subunits were f i r s t p r e c i p i t a t e d w i t h 10 mM MgCl 2, c o l l e c t e d at 2,000 rpm f o r 10 min, then heated t o 100°C i n 0.1% SDS, 10 mM T r i s pH 7.2, 20% sucrose, 0.15 M 2-mercaptoethanol before SDS g e l e l e c t r o -p h o r e s i s , ( i i i ) Subunits were p r e c i p i t a t e d w i t h MgCl 2, then p r o t e i n s were e x t r a c t e d w i t h 0.2 N HC1. Non-histone chromosomal p r o t e i n s were i s o l a t e d from whole chromatin [prepared as i n M a t e r i a l s and Methods, s e c t i o n I I ] as d e s c r i b e d by LeStourgeon and Rusch (161) , by s e q u e n t i a l treatment w i t h s a l t , a c i d , b u f f e r - s a t u r a t e d phenol (pH 8.2) and hot SDS. A l t e r n a t i v e l y , chromatin was e x t r a c t e d w i t h 0.4 N H2SOi| (removing histones) , organic s o l v e n t (removing l i p i d ) -83-and f i n a l l y w i t h 1% SDS, 0.14 M 2-mercaptoethanol, 10 mM sodium phosphate pH 7.2, t o d i s s o c i a t e non-histone p r o t e i n s , e s s e n t i a l l y as des c r i b e d by E l g i n and Bonner (162). (b) Gel e l e c t r o p h o r e s i s of p r o t e i n s Three systems were a p p l i e d to chromatin p r o t e i n s , the f i r s t two being s p e c i f i c a l l y f o r b a s i c p r o t e i n s (histones and protamines). ( i ) P r o t e i n samples were el e c t r o p h o r e s e d on urea-aluminum l a c t a t e s t a r c h g e l s as de s c r i b e d by Sung and Smithies (120) [see t h i s t h e s i s , P a r t I , M a t e r i a l s and Methods, s e c t i o n I I ( b ) ] . ( i i ) P r o t e i n samples were elect r o p h o r e s e d on urea-15% polyacrylamide g e l s as des c r i b e d by Panyim and C h a l k l e y (157). The f o l l o w i n g volumes of stock s o l u t i o n s : - 1 ml TEMED s o l u t i o n (48 ml N KOH, 17.2 ml a c e t i c a c i d , 4 ml TEMED, 30 ml of H 2 O ) , 2 ml acrylamide s o l u t i o n (60 g acrylamide, 0.4 g N,N'-methylenebisacrylamide i n 100 ml H 2 O ) and 5 ml p e r s u l f a t e - u r e a s o l u t i o n (0.2% ammonium p e r s u l f a t e i n 10 M urea) - were combined and polymerized i n g e l tubes under 3 M urea. P r o t e i n (10-50 ug) was loaded i n 6 M urea w i t h methyl green as a marker, and electr o p h o r e s e d i n 3-alanine t r a y b u f f e r (31.2 g 3-alanine, 8 ml a c e t i c a c i d d i l u t e d t o 1000 ml w i t h H 2 O ) at 4-5 millamperes per g e l f o r 1-1% hours. Gels were s t a i n e d overnight w i t h Coomassie B r i l l i a n t Blue -84-(Serva, Germany) i n m e t h a n o l : a c e t i c acid:water, 5:1:5, and d e s t a i n e d i n the same s o l v e n t . ( i i i ) The 0.1% SDS 5% p o l y a c r y l a m i d e g e l s of Weber and Osborn (163) were used to analyse p r o t e i n content. The f o l l o w -i n g volumes of stock s o l u t i o n s : - 4.5 ml of I (22.2 g a c r y l a -mide, 0.6 g IS^N'-methylenebisacrylamide i n 100 ml of H 2 O ) , 1.0 ml of I I (30 mg ammonium p e r s u l f a t e f r e s h l y d i s s o l v e d i n 2 ml H 2 O ) and 10.0 ml of I I I (28.25 ml M NaH 2P0^, 145 ml 0.5 M N a 2 H P O 4 , 10 ml o f 10% SDS, 317 ml o f H 20) - were com-bined w i t h 15 y l o f TEMED and 4.5 ml of H 20, and polymerized i n tubes under i s o b u t a n o l . P r o t e i n samples were u s u a l l y heated i n 1% SDS, 10 mM T r i s or sodium phosphate pH 7.2, 10% sucrose or g l y c e r o l and 0.002%-bromophenol b l u e , then e l e c -t r o p h o r e s e d i n t r a y b u f f e r (0.1 M phosphate, 0.1% SDS made by 1:1 d i l u t i o n of stock s o l u t i o n I I I w i t h H 20) a t 7-8 m i l l i a m p e r e s per tube f o r 4-5 hours. Gels were s t a i n e d w i t h Coomassie Blue and d e s t a i n e d as d e s c r i b e d above. (c) Q u a n t i t a t i o n of p r o t e i n s (i) By Absorbance a t 230 nm. H i s t o n e standards at v a r y -i n g c o n c e n t r a t i o n s show an absorbance, of 3.5 A 2 3 0 per 1 mg/ml. For protamines, 1 A 2 3 0 - 1 mg/ml. ( i i ) T r i c h l o r o a c e t i c a c i d t u r b i d i t y . In t h i s method of Bonner e t a l . (164), 0.2 ml of h i s t o n e sample i s combined w i t h 0.6 ml H 2 O and 0.4 ml of 50% t r i c h l o r o a c e t i c a c i d (3.3 M). -85-The r e s u l t i n g t u r b i d i t y i s monitored a t 400 nm 13-15 min a f t e r the r e a c t i o n was s t a r t e d . Standard s o l u t i o n s o f h i s -tone are done c o n c u r r e n t l y . 1 At,0o - 107.5 yg/ml o f h i s t o n e . ( i i i ) From p o l y a c r y l a m i d e g e l scans. Urea-15% p o l y a c r y l a -mide o r SDS-5% p o l y a c r y l a m i d e g e l s , s t a i n e d w i t h Coomassie Blue, were scanned w i t h a G i l f o r d spectrophotometer and the areas under each scanned p r o t e i n peak were measured. Dye b i n d i n g was p r o p o r t i o n a l to amount of p r o t e i n p r e s e n t , a l -though c o r r e c t i o n s were nec e s s a r y i n comparing p r o t e i n s of d i f f e r e n t m o l e c u l a r weight (bigger p r o t e i n s b i n d more dye ) . RESULTS I. C h a r a c t e r i z a t i o n o f DNA Fragments Produced by M i c r o -c o c c a l Nuclease D i g e s t i o n In 1973, Hewish and Burgoyne (146) r e p o r t e d t h a t d i g e s -t i o n of DNA i n r a t l i v e r n u c l e i by an endogenous nu c l e a s e produced, not the expected smear o f DNA fragments o f a l l s i z e s , but r a t h e r DNA fragments which were i n t e g r a l m u l t i p l e s of a u n i t l e n g t h . N o l l (147) confirmed t h i s r e s u l t and showed t h a t m i c r o c o c c a l n u c l e a s e gave s i m i l a r d i g e s t i o n p a t t e r n s . N u c l e i t r e a t e d w i t h t h i s n u c l e a s e y i e l d e d DNA o f l e n g t h s 200, 400, 600 e t c . base p a i r s . These r e s u l t s s t r o n g -l y suggested t h a t the arrangement o f p r o t e i n s on DNA, f a r from b e i n g random, was an ordered r e p e t i t i v e one every 200 base p a i r s , w i t h n u c l e a s e - s e n s i t i v e r e g i o n s between. We were f i r s t i n t e r e s t e d i n l o o k i n g a t m i c r o c o c c a l n u c l e a s e d i g e s t e d DNA i n t r o u t t e s t i s a t e a r l y stages o f development. Chromatin from such t e s t e s i s s i m i l a r i n p r o -t e i n c o m p o s i t i o n (and c o n t a i n s l i t t l e m e i o t i c t i s s u e , and no protamines) and p r o p e r t i e s t o chromatin i n the r a t l i v e r n u c l e i used by Hewish and Burgoyne, and N o l l . (a) DNA fragments from d i g e s t e d n u c l e i When e a r l y n u c l e o h i s t o n e stage t e s t i s n u c l e i are d i g e s -t e d w i t h m i c r o c o c c a l n u c l e a s e , the DNA i s reduced t o d i s -c r e t e fragments when a n a l y z e d on 2.5% p o l y a c r y l a m i d e g e l s -87-(Fig. l a ) . Extended digestion of such n u c l e i converts great-er than 80% of the DNA to fragments corresponding to the smallest unit length (Fig. l b ) . N o l l has c a l i b r a t e d the m o b i l i t i e s of such DNA fragments (from r a t l i v e r nuclei) with the same 2.5% polyacrylamide gel system against sequenced DNA markers (147). He provided good evidence that the fr a g -ments so produced are 205 ± 15 base pairs long, and multiples (approximately 400, 600 etc. base pairs) thereof. A p l o t of log DNA band number vs square root of band mobility for these fragments was l i n e a r (147), where the smallest fragment i s band 1 (monomer), the next largest i s band 2 (dimer) etc. A p l o t of log DNA band number vs square root of mobility i s also l i n e a r f o r the DNA fragments from trout t e s t i s (Fig. 2), i n d i c a t i n g that the larger DNA fragments are approximately i n t e g r a l multiples i n length of the smallest DNA monomer piece. We estimate a single-stranded length of roughly 200 ± 30 nucleotides for t h i s trout monomer DNA piece, using poly-acrylamide gel electrophoresis i n 99% formamide (Fig. 3) (160) against Drosophila 5S and tRNA markers, and based on Noll's c a r e f u l c a l i b r a t i o n of RNA and DNA m o b i l i t i e s i n t h i s system (154) (Fig. 4) . Closer examination of the smallest trout DNA fragment shows that i t consists of two species corresponding to monomer, 200 ± 30 base p a i r s , and a smaller, p a r t i a l l y cleaved monomer DNA, 175 + 25 base pairs (Fig. 5a, bands 1 -88-b i i i i i i 0 20 4.0 6.0 Distance, cm. FIG. 1. 2.5% polyacrylamide g e l scans of DNA: (a) from h i s -tone stage n u c l e i d i g e s t e d w i t h 100 A 2 6 0 u n i t s / m l of micro-c o c c a l nuclease, 3 min (b) from h i s t o n e stage n u c l e i d i g e s t e d w i t h 600 u n i t s / m l of m i c r o c o c c a l nuclease, 6 min (c) from l a t e protamine stage n u c l e i d i g e s t e d w i t h 600 u n i t s / m l of mi c r o c o c c a l nuclease, 6 min. DNA was prepared from n u c l e i by phenol e x t r a c t i o n , and was electrophoresed on Loening (159) 2.5% g e l s as des c r i b e d i n Methods. Gels were s t a i n e d i n S t a i n s a l l , destained i n l i g h t and scanned a t 550 nm i n a G i l f o r d spectrophotometer. -89-FIG. 2. 2.5% p o l y a c r y l a m i d e g e l s of DNA fragments from d i g e s -t e d n u c l e i . P l o t of DNA band number a g a i n s t square r o o t o f band m o b i l i t y . Data i s taken from the g e l i n F i g u r e l a . -90-FIG. 3. Denaturing 99% formamide, 6% polyacrylamide g e l separation of DNA fragments. DNA, prepared by phenol e x t r a c t i o n from n u c l e i , was d i s s o l v e d i n b u f f e r e d formamide, heated to 70°C and e l e c -trophoresed on 6% gels as i n Methods, according to the pro-cedure of Staynor et a l . (160). Gels were s t a i n e d i n S t a i n s a l l and l i g h t d estained. 5 10 Distance migrated FIG. 4. C a l i b r a t i o n o f m o b i l i t y vs s i n g l e stranded l e n g t h f o r n u c l e i c a c i d s on d e n a t u r i n g 99% formamide, 6% p o l y a c r y l a m i d e g e l s a c c o r d i n g t o N o l l (154). The r e l a t i v e m o b i l i t i e s o f . D r o s o p h i l a 5S RNA f _ A ) , tRNA ( • ), t e s t i s monomer ( [JIT ) and trimmed monomer (|o |) were determined from a formamide g e l and superimposed on N o l l ' s c a l i b r a t i o n c urve. The boxes are meant to i n d i c a t e the e r r o r s p o s s i b l e (=15%) i n such an e s t i m a t i o n . -92-and l a ) . N o l l has estimated a length of 170 + 10 base pairs for t h i s cleaved monomer (147) from r a t l i v e r n u c l e i . This smaller monomer probably derives, as suggested by N o l l (147), from endonucleolytic cleavage i n part of the 200 base pairs protected by protein, r e s u l t i n g i n a "trimming" or removal of a more nuclease accessible 30-35 base p a i r spacer region between 170 base p a i r long protein-protected regions. On 2.5% gels which have been overloaded with monomer DNA, two fragments even smaller than 170 base pairs appear (Fig. 6) but i n extremely low y i e l d compared with the un-fractionated DNA sample (not overloaded) shown i n F i g . 6. These two small fragments again probably represent i n t e r n a l endonuclease cleavages at less accessible s i t e s i n the pro-tein-protected DNA regions. (b) DNA fragments from digested chromatin When i s o l a t e d chromatin, as opposed to i n t a c t n u c l e i , i s digested with micrococcal nuclease for 1-10 minutes, the same DNA fragments can be i s o l a t e d and characterized on 2.5% polyacrylamide gels (Fig. 5b) or as single-stranded fragments on 99% formamide, 6% polyacrylamide gels. C l e a r l y here (Fig. 5b) the small DNA fragment consists of 2 species, monomer (band 1) and "trimmed" monomer (band l a ) . The recovery of such d i s c r e t e fragments from micrococcal nuclease digests further support the model (14 6-154) that 0.8 0.4 O io 0.8 0.4 -93-b 3 2 I I 1 1a BAND NO. 0 3.0 6.0 Distance migrated, cm. FIG. 5. C h a r a c t e r i z a t i o n of DNA fragments (a) from i n t a c t n u c l e i and (b) from i s o l a t e d chromatin, d i g e s t e d w i t h m i c r o -c o c c a l n u c l e a s e . I n t a c t n u c l e i (a) or i s o l a t e d chromatin (b) were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e under i d e n t i c a l con-d i t i o n s as d e s c r i b e d i n M a t e r i a l s and Methods. DNA was p r e -pared by phenol e x t r a c t i o n and e t h a n o l p r e c i p i t a t i o n , and an a l y s e d on 2.5% p o l y a c r y l a m i d e g e l s . Gels were s t a i n e d i n " S t a i n s a l l " , and scanned a t 550 nm i n a G i l f o r d s p e c t r o p h o t o -meter. -94-monomer, bands 1,1a minor bands FIG. 6. 2.5% polyacrylamide gel of DNA extracted from chrom a t i n subunit monomers. DNA was phenol extracted, e l e c t r o -phoresed as for F i g . 1 then stained with S t a i n s a l l and de-stained i n l i g h t . Note the 2 DNA bands smaller than monomer band 1. -95-chromatin ( i n s i t u i . e . i n n u c l e i , or i s o l a t e d ) c o n s i s t s l a r g e l y of 200 base p a i r long segments of DNA, covered w i t h p r o t e i n s , p a r t of which i n c l u d e s a sm a l l spacer r e g i o n of DNA (30-35 base p a i r s ) between su b u n i t s , which i s a c c e s s i b l e to nuclease d i g e s t i o n . I I . P r e p a r a t i o n of I s o l a t e d Chromatin Subunits In a d d i t i o n t o c h a r a c t e r i z i n g DNA fragments produced by m i c r o c o c c a l nuclease d i g e s t i o n , N o l l succeeded i n separ-a t i n g chromatin from n u c l e a s e - t r e a t e d n u c l e i i n t o 11.2S "subunit monomers" ( c o n t a i n i n g chromosomal p r o t e i n s on 200 base p a i r s of DNA) and l a r g e r oligomers (147). This s e c t i o n d e t a i l s the p r e p a r a t i o n and s i z e d e t e r m i n a t i o n of chromatin subunits from t r o u t t e s t i s . (a) Sucrose gradient p r e p a r a t i o n of chromatin subunits Chromatin was i s o l a t e d i n 0.2 mM EDTA from n u c l e i d i -gested b r i e f l y (2 min) w i t h 500 U/ml m i c r o c o c c a l nuclease, and was f r a c t i o n a t e d on a 10-30% sucrose g r a d i e n t . The r e -s u l t a n t A 2 6 0 p r o f i l e ( F i g . 7) shows a main peak (at = 11S), a second peak (at = 16S) and a shoulder of heavier m a t e r i a l . C h a r a c t e r i z a t i o n on 2.5% polyacrylamide g e l s of the DNA e x t r a c t e d from f r a c t i o n s of the g r a d i e n t (photos i n F i g . 7 ) , shows t h a t the main 11S peak c o n t a i n s monomer DNA, w h i l e the second peak contains predominantly dimer DNA, and the shoulder r e g i o n has even l a r g e r DNA oligomers. Note again -96-8 16 24 FRACTIONS FROM TOP, 0.4ml FIG. 7. C h a r a c t e r i z a t i o n of DNA i n chromatin subunits from d i g e s t e d n u c l e i . Chromatin was i s o l a t e d from n u c l e i d i g e s t e d w i t h m i c r o c o c c a l nuclease, and f r a c t i o n a t e d on a 5-30% sucrose g r a d i e n t as described i n M a t e r i a l s and Methods. M a t e r i a l from the g r a d i e n t f r a c t i o n s was p r e c i p i t a t e d w i t h 10 mM Mg + +, resus-pended i n 1% SDS, 20 mM EDTA, 1 M NaCl and e x t r a c t e d w i t h phenol. DNA was p r e c i p i t a t e d w i t h 2 volumes of ethanol and analysed on 2.5% polyacryalmide g e l s . Gels were s t a i n e d w i t h " S t a i n s a l l " ; g e l photos appear above corresponding g r a d i e n t f r a c t i o n s . -97-the 2 bands sm a l l e r than 170-200 base p a i r s long which appear on overloaded DNA g e l s (but i n very low y i e l d , judging from the u n f r a c t i o n a t e d chromatin sample). (b) Sedimentation v e l o c i t y a n a l y s i s of chromatin subunits As noted i n M a t e r i a l s and Methods, a s o l u t i o n of chromatin subunits was sedimented i n the Model E u l t r a e e n t r i f u g e , and the moving boundary was monitored w i t h time by d i r e c t scanning at 260 nm or by S c h l i e r e n o p t i c s . Table 1 shows the d i s p l a c e -ment vs time data f o r (a) 0.8 A2 6o/ml subunits by d i r e c t scanning, and (b) 60 OD/ml of subunits using S c h l i e r e n o p t i c s . The slopes o f l o g (displacement) vs time p l o t s are used t o c a l c u l a t e the s value as f o l l o w s : r e c a l l In r / r o = w 2s (t - to) , where t i s i n minutes l o g i o r / r o = 2^3Q3S ^ ~ t o ^ ' w ^ e r e t i s i n So the slope of such a p l o t = seconds 60 w 2s 2.303 AND 2.303 ,., . 1 0 rpm s = -gQ— (slope) -pr , w = 2* ^ - . Co r r e c t i o n s are then made f o r the d i f f e r e n t d e n s i t y (p) and v i s c o s i t y (n) of 20 mM T r i s - H C l at 4°C as compared t o water at 20°C, [P20,w/p = 1.005, n / n 2 o , w = 1.52] assuming V, the p a r t i a l s p e c i f i c volume f o r chromatin, i s 0.69 (149). Whereas d i r e c t scanning gives i n f o r m a t i o n only on the slowest (- 11S) component, using S c h l i e r e n o p t i c s , chromatin - 9 8 -TABLE I Chromatin S u b u n i t s - S e d i m e n t a t i o n V e l o c i t y Data (a) Scanner O p t i c s 36,000 rpm; 20°C; 20 mM T r i s pH 7.6 Time (t) Displacement (r) L o g i o r 0 min 5.978 cm 0.7766 18 6.1063 0.7855 32 6.2029 0.7926 45 6.2995 0.7993 62 6.3639 0.8038 78 6.5249 0.8145 s 2 0 # w = s l o p e x — 4.38 x 10 " .303/60) " x 2.701 x 1 0 " 9 x 1.005 = 11.8S TABLE I (b) S c h l i e r e n O p t i c s 40,000 rpm; 5 ° C ; 20 mM T r i s pH 7.6 monomer dimer t r i m e r Time (t) D i s p l a c e m e n t (r) L o g i o r D i s p l a c e m e n t (r) Logio r D i s p l a c e m e n t (r) Logio r 16 min 5.944 cm 0.7741 - - - — 32 6.0217 0.7797 - - - -48 6.0993 0.7852 6.193 cm 0.7919 6.292 cm 0.7987 64 6.1792 0.7909 6.320 0.8007 6.452 0.8097 80 6.2616 0.7967 6.438 0.8087 6.607 0.8200 96 6.3486 0.8027 6.556 0.8166 6.767 0.8304 ID monomer i 2 o = s l o p e x ( 2 - 3 ° 3 / 6 0 > x n T , b ( l - v p ) 2 0 , w T120 /W d - ^ P ) T , b ' W * W s 3.4 x l O - * x 2.187 x 1 0 _ 9 x 1.52 x 1.005 dimer 11.3S S 2 0 / W = 4 .9 x 10 - 1* x 2.187 x 1 0 " 9 x 1.52 x 1.005 = 16.4S t r i m e r S 2 o , w = 6.6 x 10"1* x 2.187 x 10~ 9 x 1.52 x 1.005 = 22.3S -100-16 32' K k 48' 64' FIG. 8. Sedimentation v e l o c i t y a n a l y s i s of chromatin from di g e s t e d n u c l e i . Chromatin (60 A2 6o/ml of DNA) was i s o l a t e d from n u c l e i d i g e s t e d w i t h m i c r o c o c c a l nuclease, and sedimented i n a s i n g l e s e c t o r c e l l a t 36,000 rpm, 5° i n 20 mM T r i s pH 7.6, 0.2 mM EDTA, on a Beckman Model E a n a l y t i c a l U l t r a c e n -t r i f u g e using S c h l i e r e n o p t i c s . Photos were taken at 16 min-ute i n t e r v a l s during the run, and S 2o w values were c a l c u l a t e d as d e s c r i b e d i n M a t e r i a l s and Methods. The phase p l a t e angle was 50°. -101-from d i g e s t e d n u c l e i separated ( F i g . 8) i n t o s e v e r a l s p e c i e s ; a major peak at l i s (monomer), a second peak at 16S (dimer) and a t h i r d a t 22S ( t r i m e r ) . The symmetry of the peaks argues s t r o n g l y f o r homogeneity i n s i z e , i f not composition. These s i z e estimates c o r r e l a t e w e l l w i t h those obtained on sucrose g r a d i e n t s ; however, the i o n c o n c e n t r a t i o n present (20 mM Tr i s - H C l ) may have been i n s u f f i c i e n t to prevent ano-malies due t o s a l t e f f e c t s . (c) Sepharose 2B chromatography Chromatin subunits i n 0.2 mM EDTA could a l s o be roughly f r a c t i o n a t e d on a Sepharose 2B column ( F i g . 9 ) , again i n low s a l t , 5 mM T r i s - H C l pH 7.6, 0.2 mM EDTA. Monomers e l u t e d i n a p o s i t i o n corresponding t o a molecular weight of a p p r o x i -mately 400,000. This i s s l i g h t l y higher than expected f o r an 11S p a r t i c l e c o n t a i n i n g 200 base p a i r s of DNA and an approximately equal weight of h i s t o n e s . This may i n d i c a t e d e v i a t i o n of the subunits from a s p h e r i c a l shape, or a l a r g e h y d r a t i o n s h e l l f o r the presumably h i g h l y charged s u b u n i t s . : i . Trout T e s t i s Chromatin Subunit S t r u c t u r e : Comparison of Nucleohistone and Nucleoprotamine Trout t e s t i s d i f f e r s from other t i s s u e s s t u d i e d s i n c e i t undergoes a change from m i t o t i c to m e i o t i c t i s s u e w i t h sub-sequent replacement of nucleohistone by nucleoprotamine. I t was of i n t e r e s t t h e r e f o r e t o see how these changes would be r e f l e c t e d i n the DNA products of m i c r o c o c c a l nuclease d i g e s t i o n . -102-FRACTION NO., 2.0 ml FIG. 9. Sepharose 2B chromatography of chromatin i s o l a t e d from nuclease-digested n u c l e i . Chromatin (10-100 A2 6 o/ml of DNA) from nuclease-digested n u c l e i was loaded on a 2.5 x 40 cm Sepharose 2B column eq u i l i b r a t e d and run i n 0.2 mM EDTA, 5 mM T r i s pH 7.6. Fractions of 2.0 ml were c o l l e c t e d and A 2 6 0 was monitored. The approximate e l u t i o n positions of E. c o l i B-galactosidase (MW = 540,000) and bovine catalase (MW = 240,000) are marked by arrows. -103-When sperm n u c l e i , which contain only nucleoprotamine, are digested with micrococcal nuclease, no DNA fragments are produced (Fig. I c ) , with undigested DNA remaining at the gel o r i g i n . No 11S chromatin subunits could be prepared from sperm n u c l e i . We next examined trout t e s t i s at intermediate stages of development, containing both nucleohistone and nucleoprotamine. In early protamine stage t e s t i s , much of the tissue i s meiotic i n o r i g i n and spermatid c e l l s , which have j u s t started making protamine, predominate (93). The y i e l d of DNA fragments from such tiss u e i s comparable to y i e l d s from histone stage mitotic t e s t i s , suggesting that meiotic and mit o t i c chromatin have a s i m i l a r high proportion of subunit structure (Table I I ) . When mid-protamine stage n u c l e i are digested with micro-coccal nuclease, DNA fragments are produced i n an amount roughly proportional to the amount of nucleohistone i n i t i a l l y present (Table I I ) . The l i s subunits from such tiss u e con-tained only histones, with n e g l i g i b l e protamine. These r e s u l t s i n dicate that the structures of nucleoprotamine and nucleo-histone must be very d i f f e r e n t . :v. Protein Composition of Chromatin Subunits Having looked at the size and DNA content of chromatin subunits, we next examined the protein complement of these subunits. Subunits showed a protein:DNA r a t i o of approxi-mately 1.2 and an A 2 3 o : A 2 5 8 r a t i o of 0.72, both lower than -104-TABLE I I Developmental stage % Nucleo-h i s t o n e % Nucleo-protamine % DNA r e l e a s e d i n t o fragments Histone 100 -• 8 0 e a r l y protamine 95 5 80 mid protamine 35 65 28 l a t e protamine 5 95 2 mature sperm - 100' 1 The % fragmented DNA from d i g e s t e d n u c l e i was determined from 2.5% p o l y a c r y l a m i d e g e l s o f e x t r a c t e d DNA. A l t e r n -a t i v e l y , chromatin s u b u n i t s were prepared and DNA determined by the diphenylamine r e a c t i o n . H i s t o n e s and protamine were q u a n t i t a t e d from 15% u r e a - p o l y a c r y l a m i d e g e l s . -105-values (1.5 and 0.8 respectively) reported for nuclease d i -gests of i s o l a t e d chromatin (149). The f i v e major histone species, as well as the t r o u t -s p e c i f i c histone H6, could be i d e n t i f i e d on gels from un-fractionated or fractionated subunits. These histones of chromatin subunits were present i n roughly the same propor-tions as i n whole chromatin i . e . roughly equimolar, as judged by polyacrylamide (157) and starch gel (120) electrophoresis. Across a sucrose gradient f r a c t i o n a t i o n of chromatin subunits, however, the monomer peak was r e l a t i v e l y d e f i c i e n t i n histone I (Fig. 10), with an excess of free histone I appearing at the top of the gradient. The histone I / t o t a l histone r a t i o of 0.2 obtained from Coomassie Blue s t a i n i n g on gels i s somewhat high, since the larger histone I binds proportionately more dye. N o l l had reported (147) the presence of non-histone proteins on chromatin subunits from r a t l i v e r n u c l e i . Such proteins have been reported i n amounts up to equal weight with the histones and DNA. As there i s great i n t e r e s t i n these proteins as possible gene regulators, we looked for non-histone proteins i n t e s t i s chromatin subunits. No non-histone proteins (> 25,000 molecular weight) were detectable by staining techniques on SDS-polyacrylamide gels, even when - 1 0 6 -0 8 16 24 FRACTIONS FROM TOP, 0.4ml FIG. 10. Histone content i n chromatin from d i g e s t e d n u c l e i , f r a c t i o n a t e d on a sucrose g r a d i e n t . Chromatin was i s o l a t e d from n u c l e i d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e , and was f r a c -t i o n a t e d on a 5-30% sucrose g r a d i e n t . Chromatin was monitored by i t s absorbance a t 260 nm. P r o t e i n s were e x t r a c t e d i n 2% SDS from M g + + p r e c i p i t a t e s o f g r a d i e n t f r a c t i o n s and e l e c t r o -phoresed on SDS 5% p o l y a c r y l a m i d e g e l s . Gels were s t a i n e d w i t h Coomassie Blue and scanned a t 550 nm on a G i l f o r d spectrophotometer. Histone I and t o t a l h i s t o n e were q u a n t i -t a t e d from scan a r e a s . -107-subunits were e x t r a c t e d w i t h 5% SDS and g e l s h e a v i l y over-loaded f o r h i s t o n e s . To e x p l a i n t h i s r e s u l t , e i t h e r - ( i ) other r e g i o n s of DNA, which are not covered by h i s t o n e s i n a subunit s t r u c t u r e , c o n t a i n a l l the non-histone p r o t e i n s , or ( i i ) t r o u t t e s t i s does not c o n t a i n non-histone p r o t e i n s i n the q u a n t i t i e s r e p o r t e d f o r other t i s s u e s . To d i s t i n g u i s h the two p o s s i b i l i t i e s , n u c l e i or i s o l a t e d chromatin were prepared and the non-histone p r o t e i n s were e x t r a c t e d w i t h 1% SDS from chromatin f o l l o w i n g HC1 and o r g a n i c s o l v e n t treatment (162) . Such non-histone p r o t e i n s make up l e s s than 5 % of the h i s t o n e s by weight i n t h i s t i s s u e , even i n those e a r l y stages where the s t r u c t u r e of chromatin and c e l l u l a r events (DNA & h i s t o n e s y n t h e s i s , m i t o s i s ) are otherwise s i m i l a r t o those of other organisms. V. The Behaviour of Chromatin Subunits i n S a l t I t was n o t i c e d f a i r l y e a r l y t h a t s u b u n i t s , l i k e i s o l a t e d chromatin, c o u l d be caused t o p r e c i p i t a t e by a d d i t i o n of Mg (10 mM). This was used to concentrate samples f o r b i o c h e m i c a l a n a l y s i s . F i g u r e 11 shows the p r e c i p i t a t i o n of subunits as assayed by t u r b i d i t y (Ai*4o) o r decrease i n A 2 6 0 ( a f t e r cen-t r i f u g a t i o n at 1,500 g, 10 min) f o r (a) monovalent (Na , K , T r i s ) and (b) d i v a l e n t (Mg 2 +, C a 2 + ) c a t i o n s . -108-* * • - ' 0 2 6 10 CaCI2, rnM FIG. 11. P r e c i p i t a t i o n of chromatin subunits by monovalent and d i v a l e n t c a t i o n s . Chromatin subunits (1.5 A2 6o/ml) were t r e a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of (a) NaCl, T r i s - H C l or (b) CaCl2, MgCl 2 and t u r b i d i t y a t 440 nm was monitored. Then samples were c e n t r i f u g e d at 4000 rpm, 10 minutes and super-natant absorbance a t 260 nm was measured. -109-Monovalent c a t i o n s (Na-1", K T, T r i s ) e f f e c t e d 50% p r e c i p i -t a t i o n o f s u b u n i t s a t c o n c e n t r a t i o n s around 7 0 mM, w h i l e d i v a l e n t c a t i o n s (Mg , Ca ) p r e c i p i t a t e d 50% o f the sub-u n i t s near 1 mM ( F i g . 5 ) , C a + + b e i n g s l i g h t l y more e f f e c t i v e than Mg + +. I t was noteworthy t h a t a c r o s s a sucrose g r a d i e n t o f sub-u n i t s , Mg* p e l l e t s o f the monomer f r a c t i o n s ( F i g . 11, f r a c -t i o n s 8-10) were t h i n and c l e a r , whereas the oligomer f r a c -t i o n s gave opaque white p e l l e t s . The d i f f e r e n t appearance o f monomer p e l l e t s c o r r e l a t e s w e l l w i t h the r e l a t i v e absence o f h i s t o n e I i n these f r a c t i o n s . -110-DISCUSSION This thesis supports the current picture of chromatin as "beads on a s t r i n g " , i . e . segments of DNA associated with c l u s t e r s of histones and separated by exposed spacer DNA. DNA Digestion Patterns Our r e s u l t s show that micrococcal nuclease digestion of i n t a c t trout t e s t i s n u c l e i gives r i s e to approximately 200 base p a i r long fragments of DNA (monomer) and multiples thereof (=400, 600 e t c . ) , as had been noted by Hewish and Burgoyne (146), N o i l (147) and Louie (148). The u n i v e r s a l i t y of t h i s r e s u l t [described i n r a t (146), mouse (148), polyoma (148), b i r d erythrocyte (166), c a l f thymus (7), yeast (7), trout t e s t i s and other organisms*] i s becoming c l e a r . One would not expect great differences i n t h i s monomer DNA s i z e , given the s i m i l a r i t y i n histone composition and chromatin properties across the phylogenetic tree. Van Holde and coworkers (7) have reported that monomer DNA i s 135-150 base p a i r s long. This may be due to more "trimming" of DNA from the monomer since f o r c a l f thymus the dimer and trimer bands c l o s e l y approximate the sizes reported by N o l l (147). There may also be differences i n c a l i b r a t i o n of the sizes of DNA fragments between the two research groups. * Reported at the EMBO Symposium on Developmental Genetics, Heidelberg, May 1975 - I l l -A l l these r e s u l t s p o i n t t o an or d e r e d r e p e t i t i v e a r range-ment of p r o t e i n s on 170 base p a i r s o f DNA, i n u n i t s s e p a r a t e d by n u c l e a s e - s e n s i t i v e DNA "spacer" r e g i o n s o f about 35 base p a i r s . We o b t a i n DNA fragments s m a l l e r than about 170 base p a i r s l o n g ( F i g . 6) o n l y i n extremely low y i e l d a f t e r d i g e s t i o n o f n u c l e i . These a p p a r e n t l y r e f l e c t c leavage a t a d d i t i o n a l s i t e s w i t h i n , as opposed t o between chromatin s u b u n i t s . C l a r k and F e l s e n f e l d (165) and Sahasrabuddhe and Van Holde (149), u s i n g extended (up to one hour) m i c r o c o c c a l n u c l e a s e d i g e s t i o n o f i s o l a t e d chromatin, r e p o r t e d t h a t the DNA was d i g e s t e d t o 100 base p a i r l o n g fragments. Refinement o f the a n a l y s i s o f d i g e s -t i o n p r o d u c t s by A x e l e t a l . (155) and Weintraub and Van Lente (156) showed them t o be d i s c r e t e fragments 45-130 base p a i r s l o n g , s e p a r a t e d by 10-15 base p a i r i n t e r v a l s . Such fragments presumably r e f l e c t i n t e r n a l c l e a v a g e w i t h i n a chromatin sub-u n i t monomer. Indeed, Sollner-Webb, F e l s e n f e l d (166) and A x e l (167) have r e c e n t l y r e p o r t e d t h a t such d i g e s t i o n proceeds through a 185 base p a i r monomer DNA, t o a trimmed monomer (140 and l o n g e r ) , ending w i t h the p r o d u c t i o n o f 8 l i m i t DNA fragments, 50-150 base p a i r s l o n g . These fragments may r e p r e s e n t p r o t e c -t i o n o f DNA through i n t i m a t e c o n t a c t w i t h the h i s t o n e s (166, 167), p o s s i b l y v i a the N H 2 - t e r m i n i o f these p r o t e i n s (156). N o l l ' s work (154) has shown t h a t another enzyme, DNase I, d i g e s t s DNA i n t o d i s c r e t e p i e c e s from 10 t o 200 n u c l e o t i d e s l o n g a t 10 n u c l e o t i d e i n t e r v a l s . T h i s work s t r o n g l y suggests -112-t h a t the DNA i n a chromatin sub u n i t i s wound around the o u t s i d e o f a h i s t o n e c o r e , w i t h some r e p e t i t i v e s t r u c t u r a l f e a t u r e w i t h i n the s u b u n i t . As p o i n t e d out by C r i c k ^ however, such fragments might a l s o be generated from a p a r t i c l e i n which much o f the DNA i s covered by h i s t o n e s : - e.g. e s s e n t i a l l y covered / d s 10 10 10 10 40 40 10 10 10 10 base p a i r — ' ' 1 • 1 1 1 1 1 1 1 l e n g t h MM, t . l i f t cleavage p o i n t s D i g e s t i o n o f chromatin w i t h DNase I I (168) has a l s o been r e p o r t e d t o g i v e a monomer DNA and hi g h e r m u l t i p l e s , but the fragments are a p p a r e n t l y s m a l l e r and t h e i r r e l a t i o n t o micro-c o c c a l n u c l e a s e fragments i s unclear.. I s o l a t e d chromatin (as opposed t o i n t a c t n u c l e i ) from t r o u t t e s t i s g i v e s the same m i c r o c o c c a l nuclease d i g e s t i o n p r o d u c t s : monomer (200 base p a i r s ) , trimmed monomer (170) and m u l t i p l e s ( F i g . 5 ) . N o l l e t a l . (169) have r e p o r t e d t h a t such d i s c r e t e fragments are not rec o v e r e d from chromatin; however t h e i r p r e p a r a t i o n o f chromatin i n c l u d e d e x t e n s i v e s h e a r i n g procedures which would cause a smearing among DNA s i z e c l a s s e s . F e l s e n f e l d and coworkers (166,167), Oudet e t a l . (170) have s i n c e r e p o r t e d t h a t from i s o l a t e d chromatin d i s c r e t e monomer DNA can be prepared. P r o t e i n Composition and Arrangement i n Chromatin Subunit P a r t i c l e s M i c r o c o c c a l n u c l e a s e d i g e s t i o n o f i n t a c t t e s t i s n u c l e i g i v e s r i s e t o 11S chromatin p a r t i c l e s (monomers) c o n t a i n i n g ^ F.H.C. C r i c k , p e r s o n a l communication -113-approximately 200 base p a i r long fragments of DNA and assoc-i a t e d h i s t o n e s . Presumptive chromatin dimers (16S) and t r i m e r s (22S) r e s u l t i n g from incomplete d i g e s t i o n could a l s o be i s o -l a t e d , c o n t a i n i n g correspondingly longer DNA pieces ( F i g . 7,8). Evidence i s converging now t h a t such p a r t i c l e s , f i r s t r eported by Van Holde and coworkers (149) and N o l l (147) are indeed r e p r e s e n t a t i v e of chromatin s t r u c t u r e i n the nucleus. Bradbury and coworkers (171) have demonstrated, by neutron d i f f r a c t i o n s t u d i e s , t h a t the r e f l e c t i o n s a t 110, 55, 37, 27 o A observed i n X-ray s t u d i e s can be b e t t e r e x p l a i n e d , not as a s u p e r c o i l , but as a g l o b u l a r mass of p r o t e i n (accounting f o r o the 110, 37 A r e f l e c t i o n s ) a s s o c i a t e d w i t h DNA. E l e c t r o n microscopy of SV40 v i r u s m i n i chromosomes (172), and chromatin o from v a r i o u s sources (150,170,173) c l e a r l y show =100 A beads of DNA-protein along a s t r i n g of DNA. Such r e s u l t s i n d i c a t e o o t h a t =200 base p a i r s ( = 680 A) are compacted i n t o =100 A f o r a packing r a t i o of approximately 7:1. How do the p r o t e i n s and DNA i n t e r a c t t o produce such a packing r a t i o ? For t r o u t t e s t i s chromatin, from the s i z e of the DNA (200 base p a i r s =130,000 d a l t o n s ) , the S value (11S), and the protein:DNA r a t i o (1.2:1), e i g h t to nine h i s t o n e molecules would be expected per chromatin monomer. Our r e s u l t s demonstrate t h a t subunit monomers ( F i g . 11) are d e f i c i e n t i n hi s t o n e H i . This r e s u l t c o r r e l a t e s w e l l w i t h the observations t h a t removal of HI r e s u l t s i n n e g l i g i b l e changes i n chromatin s t r u c t u r e , when analyzed by p h y s i c a l means (66-68). Indeed, -114-nuclease d i g e s t i o n s performed on H l - d e f i c i e n t chromatin g i v e the same r e s u l t s (148,170). The f a c t t h a t the h i s t o n e s appear t o be roughly equimolar i n chromatin s u b u n i t s would argue f o r the presence of two of each of the major h i s t o n e s (except h i s t o n e I) as was suggested by Kornberg (152). H e t e r o g e n e i t y w i t h r e s p e c t t o which s p e c i e s of h i s t o n e are p r e s e n t on i n -d i v i d u a l chromatin s u b u n i t s i s not r u l e d out however. P r o t e i n c r o s s l i n k i n g r e s u l t s would be extremely u s e f u l i n o b t a i n i n g a crude t o p o g r a p h i c a l map of the chromatin s u b u n i t , as has been done with ribosomes (22). Recently Thomas and Kornberg (174) have shown t h a t c r o s s l i n k i n g of the h i s t o n e s of i s o l a t e d chromatin s u b u n i t s proceeds up t o an octamer, which may c o n s i s t of 2 each of H2A, H2B, H.3 and H4. H2A-H2B dimers and (H3) 2(H4)2(H2A)(H2B) hexamers were a l s o observed. These h i s t o n e oligomers are s i m i l a r t o those prepared from i s o l a t e d chromatin (32-34) and l e a d to a p i c t u r e of a chromatin s u b u n i t as a g l o b u l a r c l u s t e r of 8 h i s t o n e s (perhaps an ( H 3 ) 2 ( H 4 ) 2 tetramer a s s o c i a t e d w i t h two H2A-H2B dimers) assoc-i a t e d w i t h DNA. The work of Van Lente and Weintraub (156) sug-g e s t s t h a t h i s t o n e s ' b a s i c N - t e r m i n i i n t e r a c t w i t h DNA while the non-polar r e g i o n s allow h i s t o n e - h i s t o n e i n t e r a c t i o n , i n a c c o r d w i t h the p r e d i c t i o n s made from sequence s t u d i e s (15,17, 23). Such a s u b u n i t might a l s o undergo " s e l f - a s s e m b l y " as has been d e s c r i b e d f o r ribosomes and c e r t a i n v i r u s e s . In support of such an i d e a , r e c e n t r e c o n s t i t u t i o n of chromatin subunits (to g i v e the same nuclease d i g e s t i o n p a t t e r n s ) , from a mixture of h i s t o n e s and DNA has been r e p o r t e d (155,170). -115-The l o c a t i o n and type of f o l d i n g of DNA i n a subunit are s t i l l u n c l e a r . N o l l ' s r e s u l t s w i t h DNase I (154) appear to show t h a t DNA i s on the out s i d e of a subunit. Such a p i c t u r e would a l s o a l l o w f o r i n t e r a c t i o n s of DNA w i t h other molecules (enzymes e t c . ) . C r i c k and Klug have proposed an a t t r a c t i v e model f o r the packing of DNA i n t o a subunit (175). The DNA i n t h i s model can be "kinked" a t about 100°, r e s u l t i n g i n a left-handed h e l i x of 20 base p a i r long s t r a i g h t s t r e t c h e s sep-arated by k i n k s . The nature of spacer DNA between subunits i s a l s o somewhat "fuzzy". The amount of DNA i n a subunit i s g e n e r a l l y accepted to be approximately 200 base p a i r s l o n g , i n c l u d i n g a spacer r e g i o n of about 30-40 n u c l e o t i d e s . However, there have been r e p o r t s (7,166,167) t h a t the l i m i t d i g e s t of monomer has a DNA length^" of approximately 140-150 base p a i r s ( i . e . 50-60 base p a i r s have been l o s t ) . I t may be then t h a t a f t e r i n i t i a l DNA cleavage t o produce a 200 base p a i r long fragment, a slower trimming of spacer (30-40) occurs, f o l l o w e d by d i g e s t i o n of even l e s s a c c e s s i b l e DNA t o 140-150 base p a i r s . E l e c t r o n micrographs of chromatin show t h a t the spacer DNA regions are extended, and v i s i b l e as "threads" between 2 subunit "beads" (150,170,173). F i n c h and coworkers at F.H.C. C r i c k , personal communication Reported at EMBO Symposium on Developmental Genet i c s , H e i d e l b e r g , 1975 -116-Cambridge, however, have claim e d t h a t s u b u n i t s are norm a l l y i n c o n t a c t w i t h each o t h e r ; l i n e a r spacer DNA i s an a r t i f a c t of sample p r e p a r a t i o n . H i s t o n e H i , as noted e a r l i e r , p r o b a b l y i s not nece s s a r y f o r m a i n t a i n i n g the b a s i c s u b u n i t s t r u c t u r e o f chromatin. The absence of H i from t e s t i s s u b u n i t monomers, and presence i n h i g h e r o l i g o m e r s ( F i g . 11) suggests t h a t H i may i n s t e a d have some c r o s s - l i n k i n g f u n c t i o n (69-71). T h i s h i s t o n e c o u l d c o n t a c t p r o t e i n s between i n d i v i d u a l monomers o r r e s i d e i n the DNA spacer r e g i o n s , m a i n t a i n i n g chromatin i n a condensed form. Indeed, Chambon and coworkers have r e p o r t e d (170) t h a t i n e l e c t r o n micrographs o f chromatin, s u b u n i t s w i t h H i p r e s e n t are c l o s e l y packed. The c r o s s l i n k i n g o f f r e e s u b u n i t s by h i s t o n e I may be the reason f o r the presence o f s m a l l amounts o f monomer DNA even i n the l a r g e r chromatin o l i g o m e r s ( F i g . 7 ) , a l t h o u g h t h i s may r e f l e c t a g g r e g a t i o n d u r i n g g r a d i e n t c e n t r i f u g a t i o n . H i s t o n e -h i s t o n e i n t e r a c t i o n s alone are e v i d e n t l y not s u f f i c i e n t t o h o l d the m a j o r i t y o f monomer s u b u n i t s t o g e t h e r , as noted by N o l l (147), as most monomer DNA runs i n the 11S monomer r e g i o n . The r e l a t i v e absence o f h i s t o n e I i n chromatin monomers, as opposed t o o l i g o m e r s , c o i n c i d e s w i t h the t h i n c l e a r Mg p e l l e t s from these p r e p a r a t i o n s . T h i s may r e f l e c t some sym-metry (e.g. DNA w i t h e i g h t h i s t o n e s i n a r e g u l a r s t r u c t u r e ) a l l o w i n g an ordered p a c k i n g o f monomers. These monomer p e l l e t s may prove u s e f u l i n p h y s i c a l s t u d i e s o f s u b u n i t s t r u c t u r e . -117-One p o s s i b l e problem might be the presence o f a t l e a s t two monomer p o p u l a t i o n s , v a r y i n g i n s i z e from those c o n t a i n i n g band 1 (=200 base p a i r s ) t o those c o n t a i n i n g band l a (=170 base p a i r s ) o r s m a l l e r DNA. F u r t h e r treatment (such as l i m i t e d exonuclease d i g e s t i o n ) might g i v e a more homogeneous p o p u l a t i o n of monomer f o r x-ray and o t h e r s t u d i e s . The presence of h i s t o n e I i n oligomers may c o n f e r asymmetry and r e s u l t i n a d i s o r d e r e d complex i n the presence of Mg + +. N o l l (147) had r e p o r t e d t h a t non-histone p r o t e i n s (NHP) were p r e s e n t i n 11S chromatin s u b u n i t s from r a t l i v e r n u c l e i . However, we c o u l d f i n d no d e t e c t a b l e non-histone p r o t e i n s i n t r o u t t e s t i s chromatin e i t h e r by the methods of LeStourgeon and Rusch (161), E l g i n and Bonner (162), o r by d i r e c t hot 5% SDS e x t r a c t i o n . A few v e r y minor p r o t e i n s p e c i e s c o u l d be ob-served i f washed n u c l e i , r a t h e r than chromatin, were used as s t a r t i n g m a t e r i a l . I t seems l i k e l y then, t h a t the non-histone p r o t e i n s observed by some workers a t t o t a l l e v e l s approaching equal weight w i t h the h i s t o n e s (135), c o u l d be n u c l e a r p r o t e i n s not a s s o c i a t e d w i t h chromatin, e.g. from membranes (137), o r c y t o p l a s m i c contaminants (138). A l t e r n a t i v e l y , the s p e c i a l n a ture o f t r o u t t e s t i s — a t i s s u e " s h u t t i n g down" g e n e t i c f u n c t i o n s f o r packaging o f DNA and protamine i n t o sperm — may account f o r the l a c k o f o b s e r v a b l e non-histone p r o t e i n s . T h i s seems u n l i k e l y however f o r the e a r l y stages o f t e s t i s development, when DNA, RNA, p r o t e i n s y n t h e s i s and c e l l d i v i s i o n are s t i l l ongoing p r o c e s s e s (93) , and the s t r u c t u r e and f u n c --118-t i o n of chromatin are s i m i l a r t o o t h e r t i s s u e s . I n s t e a d , the l a r g e n u c l e a r / c y t o p l a s m i c r a t i o makes i t r e l a t i v e l y easy t o prepare chromatin f r e e of contaminants; t h i s may account f o r the low l e v e l s of NHP p r e s e n t . C e r t a i n l y some p r o t e i n s l i k e a c t i n , myosin (176) and n u c l e a r enzymes must be p r e s e n t ; however, i t seems l i k e l y t h a t any p r o t e i n s i n v o l v e d i n the s p e c i f i c r e g u l a t i o n o f gene e x p r e s s i o n i . e . NHP, need not be p r e s e n t i n the l a r g e amounts r e p o r t e d by some workers. Chromatin S t r u c t u r e During T e s t i s M a t u r a t i o n A t e a r l y p r e - m e i o t i c stages of t e s t i s development, i t s chromatin has the same s u b u n i t s t r u c t u r e as t h a t r e p o r t e d f o r o t h e r organisms. Our r e s u l t s i n d i c a t e f u r t h e r t h a t the n u c l e o -h i s t o n e o f m e i o t i c t i s s u e has a s i m i l a r s u b u n i t s t r u c t u r e . Such r e s u l t s a r e an average a c r o s s c e l l s i n d i f f e r e n t stages of the c e l l c y c l e . I t may be f o r example t h a t the s m a l l p r o -p o r t i o n of c e l l s undergoing c e l l d i v i s i o n would show d i f f e r -ences i n s u b u n i t s t r u c t u r e . Such d i f f e r e n c e s have been ob-served f o r c e l l s a r r e s t e d i n metaphase; the h i g h l y condensed chromosomes a p p a r e n t l y g i v e d i f f e r e n t , l a r g e r DNA fragments upon m i c r o c o c c a l n u c l e a s e d i g e s t i o n ^ . Nucleoprotamine was not d i g e s t e d to produce DNA fragments analogous to those f o r n u c l e o h i s t o n e . Protamine was not simply an i n h i b i t o r of the n u c l e a s e because i n t i s s u e c o n t a i n i n g both h i s t o n e and protamine, s u b u n i t s c o u l d be r e c o v e r e d i n F.H.C. C r i c k , p e r s o n a l communication -119-amounts p r o p o r t i o n a l t o the amount of h i s t o n e p r e s e n t (Table I I ) . Nucleoprotamine may not then have a s u b u n i t s t r u c t u r e analogous to n u c l e o h i s t o n e , but i n s t e a d e i t h e r ( i ) a l l DNA i s covered w i t h protamine, o r ( i i ) i f a s u b u n i t - l i k e s t r u c t u r e i s p r e s e n t , spacer DNA i n t i g h t l y condensed nucleoprotamine may be s t e r i c a l l y i n a c c e s s i b l e . T h i s t i g h t l y condensed s t r u c -t u r e o f nucleoprotamine has been noted from o t h e r s t u d i e s . For example, when chromatin c o n t a i n i n g both n u c l e o h i s t o n e and nucleoprotamine i s sheared, o n l y n u c l e o h i s t o n e i s s o l u b i l i z e d i n t o s m a l l e r fragments (177). I t i s e v i d e n t then t h a t t h e r e i s something unique about the p a c k i n g o f DNA w i t h protamine i n t o t h e s m a l l volume o f a sperm head. The s t r u c t u r e o f t h i s nucleoprotamine might be seen v i a o t h e r n u c l e a s e s e.g. DNase I I . One i n t e r e s t i n g problem i s the s t r u c t u r e o f n u c l e o h i s t o n e i n those sperm c e l l s (e.g. g o l d f i s h ) which do not c o n t a i n protamine. I t remains t o be seen whether o t h e r mechanisms o f c o n d e n s a t i o n i n such sperm c e l l s w i l l render the chromatin i n a c c e s s i b l e t o m i c r o c o c c a l n u c l e a s e . C o n c l u s i o n The a v a i l a b i l i t y o f l a r g e amounts o f r e a d i l y prepared n u c l e i from t r o u t t e s t i s has g r e a t l y f a c i l i t a t e d these s t u d i e s , which s t r o n g l y support the "bead on a s t r i n g " p i c t u r e o f chromatin. We now have a c o n c e p t u a l framework around which t o p l a n f u r t h e r s t u d i e s on: -120-(i) the nature of the DNA (85%) complexed with protein, and that DNA (the other 15%) which i s digested by nuclease i . e . in which kind of DNA.are the "active" genes? One approach to t h i s question i s v i a h y b r i d i z a t i o n of s p e c i f i c RNA t r a n s c r i p t s to subunit (histone-protected) DNA or to DNA containing spacer regions. Axel et a_l. (178) have recently reported that globin genes are p a r t i a l l y (80%) covered by proteins. They conclude that the presence of protein on DNA i s not s u f f i c i e n t to re-s t r i c t t r a n s c r i p t i o n . Similar r e s u l t s have been reported"'' for ribosomal rRNA genes, although i n t h i s case the r e s u l t s may be equivocal, because multiple copies of the rRNA genes e x i s t i . e . 99 of 100 copies may be protein-covered (and not expressed), while the 1 per 100 could be open and transcribed; ( i i ) the changes i n DNA sequences covered by histones, when new genetic information i s expressed. Are s p e c i f i c sequences covered by protein? I f genes are protein-covered, how are they recognized and activated? For SV40 v i r a l DNA there appears to be no sequence s p e c i f i c i t y for histone binding, based on the random cleavage of SV40 "minichromosomes" by r e s t r i c t i o n enzymes (179); ( i i i ) the presence of non-histone proteins (NHP) and t h e i r role i n gene expression. I f bur r e s u l t s on the r e l a t i v e ab-sence of NHP prove u n i v e r s a l , then r a d i o a c t i v e - l a b e l l i n g (with [ 3 5S]methionine, 1 2 5 I , 3 2P) may be necessary to detect these Chambon, P. et a l . , and Jones, A. and Reeves, R., unpublished r e s u l t s -121-proteins and t h e i r d i s t r i b u t i o n i n subunit monomers and higher oligomers; (iv) the location and mode of action of the enzymes responsible for gene expression (DNA & RNA polymerase, nucleases modifying enzymes, e t c . ) . For example, does micrococcal nuclease d i -gestion of spacer DNA release enzymes (RNA polymerase?) assoc-iated v/ith that spacer DNA? (v) the assembly of chromatin subunits. I t may be that the histones and DNA w i l l assemble i n v i t r o i n an ordered ser i e s of reactions, much as the ribosome does (180). The use of modi-f i e d or unmodified histones may show something about the im-portance of acetylation, phosphorylation etc. i n assembly. Such i n v i t r o r e c o n s t i t u t i o n experiments may also show how histone Hi contributes to the organization and packing of sub-units into a higher order structure. I t would also be of i n t e r e s t to see, v i a [11*C] amino acid and [ 3H]thymidine l a b e l l i n g , the time course of radioactive incorporation into subunits. One might p r e d i c t that with i n -creasing l a b e l l i n g time, [ 3H]thymidine i n acid-soluble (spacer) DNA should r i s e and plateau, while 3H i n subunit (protected) DNA would r i s e a f t e r a short lag time equal to the time re-quired for assembly. On the other hand, i f newly synthesized histone i s not properly bound to DNA u n t i l a c e t y l a t i o n and de-acetylation 12-16 hours afte r synthesis (84), then [ ^ C ] l y s i n e would not appear i n l i s subunits. [ 1 "*C] acetate would behave s i m i l a r l y , while [ 1"c]methyl l a b e l , which should occur on -122-" o l d " (subunit-bound) h i s t o n e s , would appear immediately i n s u b u n i t s . I t may even be p o s s i b l e t o se p a r a t e d i f f e r e n t p o p u l a t i o n s o f s u b u n i t s on very weak ion-exchangers, based on s m a l l d i f f e r -ences i n h i s t o n e m o d i f i c a t i o n and hence charge; (vi) the t h r e e - d i m e n s i o n a l s t r u c t u r e o f the chromatin s u b u n i t . I t seems l i k e l y t h a t c r o s s l i n k i n g s t u d i e s (174) may g i v e a good i d e a of the arrangement o f p r o t e i n s i n a s u b u n i t v e r y soon. 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